Substitutional variants of hydroxynitrile lyase with high specific activity and methods of use

ABSTRACT

An improved hydroxynitrile lyase characterized by having a mutation of substitution of at least one amino acid residue in the amino acid sequence of a wild-type hydroxynitrile lyase with another amino acid and by its hydroxynitrile lyase activity per transformant being higher than the hydroxynitrile lyase activity per transformant into which the wild-type hydroxynitrile lyase gene is introduced; and a method for producing a hydroxynitrile lyase, comprising expressing the improved hydroxynitrile lyase in a host and recovering the improved hydroxynitrile lyase from the resultant culture.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a 35 U.S.C. §371 National Stage patentapplication of International patent application PCT/JP05/019360, filedon Oct. 14, 2005, which claims priority to Japanese patent applicationJP 2005-058857, filed on Mar. 3, 2005, Japanese patent application JP2004-355766, filed on Dec. 8, 2004, and Japanese patent application JP2004-301718, filed on Oct. 15, 2004.

TECHNICAL FIELD

The present invention relates to an improved hydroxynitrile lyase and amethod for producing the improved hydroxynitrile lyase.

BACKGROUND ART

Hydroxynitrile lyase is an enzyme that catalyzes a reaction forproducing cyanohydrins. Briefly, hydroxynitrile lyase is an enzymecatalyst used in the synthesis of cyanohydrins (α-hydroxynitriles) fromcarbonyl compounds in the presence of a cyanide donor. Cyanohydrins canbe converted into various compounds and are useful as intermediates inorganic synthesis. Further, optically active cyanohydrins may be used inthe synthesis of α-hydroxyacids, α-hydroxyketones, β-aminoalcohols andthe like, and are extremely useful in the synthesis of various importantoptical intermediates which are used for producing, for example,pharmaceuticals and chemicals. Therefore, development of a method forproducing a large quantity of hydroxynitrile lyase is desired.

The hydroxynitrile lyase that catalyzes cyanohydrin synthesis isclassified into the two groups of (S) selective group and (R) selectivegroup. Of these groups, (S)-hydroxynitrile lyase catalyzes a reaction toproduce (S)-cyanohydrin from either ketone or aldehyde and a cyanidecompound under acidic conditions. As a representative reaction of thistype, a reaction may be given in which (S)-mandelonitrile is producedfrom benzaldehyde and a cyanide compound, prussic acid.(S)-Hydroxynitrile lyase is also used as a biocatalyst capable ofproducing from inexpensive substrates optically active substances highlyuseful as intermediates for pharmaceuticals and chemicals. Thus,(S)-hydroxynitrile lyase is extremely useful in many fields. In order toutilize hydroxynitrile lyase in industrial production of opticallyactive cyanohydrins, development of a method for producing a largequantity of a hydroxynitrile lyase which has high activity per cell orprotein and high stereo-selectivity is desired.

It is known that hydroxynitrile lyase exists only in those plants thathave cyanogenic glucosides. For example, as (R)-hydroxynitrile lyases,those derived from plants of the family Rosaceae, such as almond (Prunusamygdalus), are known. Further, as (S)-hydroxynitrile lyases, thosederived from plants of the family Gramineae, such as sorghum (Sorghumbicolor); plants of the family Euphorbiaceae, such as cassava (Manihotesculenta) and Para rubber tree (Hevea brasiliensis); and plants of thefamily Olacaceae, such as tallow wood (Ximenia americana); are known.However, it has only been possible to extract extremely small quantitiesof hydroxynitrile lyases from these plants.

In order to obtain a large quantity of a hydroxynitrile lyase which isuseful in pharmaceutical and chemical fields, attempts to obtain such ahydroxynitrile lyase with genetic engineering methods have beenmade⁽¹⁻⁹⁾. However, when a heterologous protein is expressed using atransformant, results obtained from researches on a correspondinghomologous protein (such as yield, biochemical activities, etc.) are notalways applicable. In other words, when a heterologous proteins isexpressed using a transformant, it is not easy to predict the behaviorand expression level of the transformant, the biochemical activities ofthe protein of interest, and so forth.

Further, depending on the type of the protein to be expressed, normalfolding of the protein may not occur in the transformed host, resultingin the formation of an inclusion body. It is known that, in many cases,the protein within this inclusion body becomes an inactive proteinwithout its inherent activity. In hydroxynitrile lyase, it is reported,for example, that 99% of hydroxynitrile lyase which has been expressedby culturing an Escherichia coli transformant at 37° C. is found in theinsoluble fraction in the form of an inactive inclusion body⁽¹⁾. It isalso reported that the enzyme activity of crude enzyme solution producedusing E. coli is 0.545 units/mg protein⁽²⁻⁴⁾; that the liquid activityof crude enzyme solution produced using E. coli (host: M15[pREP4]) is0.5 U/ml⁽¹⁴⁾; and that the specific activities of crude enzyme solutionsare 0.20 U/mg protein (host: Top10′, 28° C.) and 0.61 U/mg protein(host: XL1-blue, 22° C.), respectively^((15, 16)). However, none of theabove-mentioned enzyme activities is satisfactory.

As means to solve these problems, for example, an attempt has been madein which a hydroxynitrile lyase-expressing E. coli transformant iscultured at a low temperature to thereby inhibit the formation ofinclusion bodies and improve the yield of a hydroxynitrile lyase havingactivity⁽⁸⁾. However, this technology requires a long time forcultivation and needs to use large quantities of utility such aselectricity and cooling water for maintaining the low temperature.Considering industrial production of hydroxynitrile lyase, thesedrawbacks will increase production cost greatly.

On the other hand, due to recent advancement in recombinant DNAtechniques, it has become rather easy to prepare a mutant protein inwhich one or more amino acids constituting the original protein aredeleted, added, inserted or substituted with other amino acids. Inparticular, when the protein of interest is an enzyme, it is known thatmutants thereof acquire improvement in properties such as stability,resistance to organic solvents, thermal resistance, acid resistance,alkali resistance, substrate specificity or substrate affinity comparedto the original enzyme, depending on the sites of the amino acidresidues deleted, added, inserted or substituted and the types of theamino acids which replace those amino acids. These improvements inproperties may bring about large reduction of production cost inindustrial production utilizing enzyme reactions, through stabilizationof enzymes as catalysts, simplification of reaction steps, improvementof reaction yield and so forth. Therefore, a large number of improvedenzymes with various improved properties are now being developed.

In hydroxynitrile lyase, mutants in which one or more constituent aminoacids are deleted, added, inserted or substituted have also beenreported. For example, it is reported that a mutant hydroxynitrile lyasehas an improved affinity to aromatic aldehydes, in particular,3-phenoxybenzaldehyde^((9, 10)). However, a great increase in theproduction yield of hydroxynitrile lyase has not been achieved yet. Itis also reported that a mutant in which tryptophan at position 128 issubstituted with another amino acid and a mutant in which cysteine atposition 81 is substituted with alanine were prepared and transformedinto E. coli M15. When these E. coli M15 transformants were cultured inTB medium containing 100 μM IPTG under conditions cooled from 37° C. to20° C., some of the mutant-expressing M15 transformants exhibited ahydroxynitrile lyase activity per cell higher than that exhibited by thewild-type hydroxynitrile lyase-expressing M15 transformants⁽⁶⁾. However,according to this document, the hydroxynitrile lyase activity of thewild-type hydroxynitrile lyase-expressing M15 transformants is about ½of that of the wild-type hydroxynitrile lyase-expressing JM109transformants obtained under the same culture conditions. Thus, theeffects of mutants are not necessarily demonstrated in hosts with highexpression ability. It is also reported that substitution of glycine atposition 113 with serine in a hydroxynitrile lyase derived from asubspecies of cassava (Manihot esculenta) grown in China increased thespecific activity of the resultant mutant hydroxynitrile lyase⁽¹¹⁾.However, the amino acid at position 113 of the hydroxynitrile lyasederived from common cassava (Manihot esculenta) is serine. Thus, thisreport merely shows that this amino acid is important for hydroxynitrilelyase activity.

On the other hand, it is reported that the N-terminal methionine presentat the time of translation undergoes processing in 40% of the proteinsin E. coli cell extract⁽¹⁷⁾. This processing is catalyzed by an enzymecalled methionine aminopeptidase⁽¹⁸⁾. It is reported that whether or notan endogenous protein in E. coli cells is ready to undergo processing byaminopeptidase is decided by the type of the amino acid at position 2 ofthe protein; and that the larger the side chains of the amino acid atposition 2 is, it is more difficult for the protein to undergo theprocessing⁽¹²⁾. There is also reported an N-end rule that “the stabilityof a protein in E. coli cells is decided by the type of the N-terminusamino acid of the protein”⁽¹³⁾. According to this N-end rule, when theN-terminus of a protein is arginine, lysine, leucine, phenylalanine,tyrosine, tryptophan or the like, the stability of the protein in cellsis low and the protein is readily degraded. Based on these findings, itmay be possible to improve the stability of a protein of interest in ahost transformant by selecting at position 2 of the protein an aminoacid which has large side chains (i.e., hard to undergo the processingby methionine aminopeptidase) and which is not arginine, lysine,leucine, phenylalanine, tyrosine or tryptophan. However, theabove-described results^((12, 13)) were obtained from analysis ofendogenous proteins in hosts or some model proteins. Therefore,hydroxynitrile lyase which is a heterologous protein to a host will notnecessarily follow the above-described rule because, as mentionedearlier, when a heterologous protein is expressed using a transformant,it is very difficult to predict the behavior and expression level of thetransformant, the biochemical activities of the protein of interest,etc.

As described so far, attempts to obtain a hydroxynitrile lyase withremarkably improved properties can not be said successful. Creation ofstill more useful hydroxynitrile lyase mutants has been stronglydesired.

REFERENCES

-   (1) Japanese Unexamined Patent Publication (kohyo) No. 11-508775-   (2) Japanese Unexamined Patent Publication No. 2000-189159-   (3) Japanese Unexamined Patent Publication No. 2000-189160-   (4) Japanese Unexamined Patent Publication No. 2000-245486-   (5) Japanese Unexamined Patent Publication No. 2002-330791-   (6) International Publication WO 01/48178-   (7) Japanese Unexamined Patent Publication No. 2004-194550-   (8) Japanese Unexamined Patent Publication No. 2004-194551-   (9) Japanese Unexamined Patent Publication No. 2000-125886-   (10) Holger Buhler et al, Chembiochem. 4 (2003) 211-216-   (11) GonghongYan et al, Biotechnol. Lett. 25 (2003) 1041-1047-   (12) Ph.-Herve Hirel et al, Proc. Natl. Acad. USA 86 (1989),    8247-8251-   (13) Alexander Varshavsky, Proc. Natl. Acad. USA 93 (1996),    12142-12149-   (14) Siegfried Forster et al, Angew. Chem. Int. Ed. Engl. 35 (1996)    437-439-   (15) Meinhard Hasslacher et al, J. Biol. Chem. 271 (1996), 5884-5891-   (16) Meinhard Hasslacher et al, Protein Expression and Purification    11 (1997), 61-71-   (17) Waller, J. P. et al, J. Mol. Bio. 7 (1963), 483-496-   (18) Ben-Bassat, A. et al, J. Bacteriol. 169 (1987), 751-757

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide an improvedhydroxynitrile lyase and a method for producing the improvedhydroxynitrile lyase.

As a result of extensive and intensive researches toward the solution ofthe above-described problems, the present inventors have found that itis possible to greatly improve hydroxynitrile lyase activity pertransformant by substituting at least one amino acid residue in theamino acid sequence of a wild-type hydroxynitrile lyase with anotheramino acid. Thus, the present invention has been achieved.

The present invention relates to the following inventions.

(1) An improved hydroxynitrile lyase selected from any one of thefollowing (A) to (G):

(A) an improved hydroxynitrile lyase in which the amino acid residue atposition 2 of the amino acid sequence of a wild-type hydroxynitrilelyase is substituted with another amino acid residue;

(B) an improved hydroxynitrile lyase in which the histidine residue atposition 103 or a neighboring position thereto of the amino acidsequence of a wild-type hydroxynitrile lyase is substituted with anotheramino acid residue;

(C) an improved hydroxynitrile lyase in which at least one lysineresidue of the amino acid sequence of a wild-type hydroxynitrile lyaseis substituted with another amino acid residue;

(D) an improved hydroxynitrile lyase in which the amino acid residue atposition 2 and the histidine residue at position 103 or a neighboringposition thereto of the amino acid sequence of a wild-typehydroxynitrile lyase are substituted with other amino acid residues;

(E) an improved hydroxynitrile lyase in which the amino acid residue atposition 2 and at least one lysine residue of the amino acid sequence ofa wild-type hydroxynitrile lyase are substituted with other amino acidresidues;

(F) an improved hydroxynitrile lyase in which the histidine residue atposition 103 or a neighboring position thereto and at least one lysineresidue of the amino acid sequence of a wild-type hydroxynitrile lyaseare substituted with other amino acid residues;

(G) an improved hydroxynitrile lyase in which the amino acid residue atposition 2, the histidine residue at position 103 or a neighboringposition thereto and at least one lysine residue of the amino acidsequence of a wild-type hydroxynitrile lyase are substituted with otheramino acid residues.

(2) The improved hydroxynitrile lyase of (1) above, wherein thewild-type hydroxynitrile lyase is derived from cassava (Manihotesculenta) or Para rubber tree (Hevea brasiliensis).

(3) The improved hydroxynitrile lyase of (1) or (2) above, wherein theamino acid residue at position 2 is substituted with any amino acidselected from the group consisting of lysine, asparagine, isoleucine,arginine, glutamine, proline, threonine, tyrosine, leucine, methionine,serine and glutamic acid.(4) The improved hydroxynitrile lyase of any one of (1) to (3) above,wherein the histidine residue at position 103 or a neighboring positionthereto is substituted with an amino acid having one or both of thefollowing properties (a) and (b):

(a) an amino acid containing one or two nitrogen atoms in its molecule;

(b) a neutral amino acid.

(5) The improved hydroxynitrile lyase of any one of (1) to (4) above,wherein the histidine residue at position 103 or a neighboring positionthereto is substituted with any amino acid selected from the groupconsisting of methionine, leucine, isoleucine, valine, cysteine,glutamine, serine, threonine, alanine and tryptophan.(6) The improved hydroxynitrile lyase of any one of (1) to (5) above,wherein at least one lysine residue present in a region from positions175 to 224 of the amino acid sequence of the wild-type hydroxynitrilelyase is substituted with another amino acid.(7) The improved hydroxynitrile lyase of any one of (1) to (6) above,wherein at least one lysine residue present in a region from positions175 to 224 of the amino acid sequence of the wild-type hydroxynitrilelyase is substituted with an amino acid having one or both of thefollowing properties (a) and (b):

(a) an amino acid containing one or two nitrogen atoms in its molecule;

(b) a neutral amino acid.

(8) The improved hydroxynitrile lyase of any one of (1) to (7) above,wherein at least one lysine residue present in a region from positions175 to 224 of the amino acid sequence of the wild-type hydroxynitrilelyase is substituted with proline.

(9) The improved hydroxynitrile lyase of any one of (1) to (8) above,wherein at least one lysine residue selected from the group consistingof the lysine residues at positions 176, 199 and 224 in the amino acidsequence as shown in SEQ ID NO: 1 is substituted with another aminoacid.(10) The improved hydroxynitrile lyase of any one of (1) to (8) above,wherein at least one lysine residue selected from the group consistingof the lysine residues at positions 175, 198 and 223 in the amino acidsequence as shown in SEQ ID NO: 102 is substituted with another aminoacid.(11) An improved hydroxynitrile lyase consisting of the amino acidsequence of the improved hydroxynitrile lyase of any one of (1) to (10)above, wherein one or several amino acids other than those amino acidsat the substitution positions specified in (A) to (G) are deleted,substituted or added.(12) An improved hydroxynitrile lyase gene encoding the improvedhydroxynitrile lyase of any one of (1) to (11) above.(13) A recombinant vector comprising the improved hydroxynitrile lyasegene of (12) above.(14) A transformant obtained by introducing the recombinant vector of(13) above into a host.(15) A culture obtained by culturing the transformant of (14) above.(16) An improved hydroxynitrile lyase recovered from the culture of (15)above.(17) A method for producing an improved hydroxynitrile lyase, comprisingrecovering the improved hydroxynitrile lyase from the culture of (15)above.

(18) A method for producing a cyanohydrin, comprising treating a ketonecompound or aldehyde compound, and a cyanide compound with the culturetransformant and recovering the cyanohydrin from the treated culture.

(19) A method for producing a cyanohydrin, comprising treating a ketonecompound or an aldehyde compound and a cyanide compound with theimproved hydroxy lyase of any one of 1 to 11 and 16 above and recoveringthe cyanohydrin from the treated cultures.

(20) A method for producing a hydroxy carboxylic acid, comprisinghydrolyzing the cyanohydin obtained by the method of 18 or 19 fromabove.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing the preparation of a plant codonwild-type hydroxynitrile lyase gene in Example 1.

FIG. 2 is a schematic diagram showing the preparation of an E. colicodon wild-type hydroxynitrile lyase gene in Example 3.

FIG. 3 is a diagram showing the comparative analysis by SDS-PAGE ofexpression levels of hydroxynitrile lyase protein in an emptyvector-introduced transformant (JM109/pKK233-2(+Sse)); a wild-typehydroxynitrile lyase-expressing transformant (JM109/pOXN103); andhydroxynitrile lyase-expressing transformants in which the 2nd aminoacid is substituted (pOXN103V2K, pOXN103V2N, pOXN103V2I, pOXN103V2R,pOXN103V2Q, pOXN103V2P, pOXN103V2T, pOXN103V2Y, pOXN103V2L, pOXN103V2M,pOXN103V2S, pOXN103V2E, pOXN103V2A, pOXN103V2G and pOXN103V2D) inExample 6. The arrow mark indicates the band of hydroxynitrile lyaseprotein.

FIG. 4 is a diagram showing the results of SDS-PAGE analysis on cellextract soluble fractions (S) and insoluble fractions (I) preparedrespectively from random mutant-expressing transformants (19-E8 and36-E10) obtained from the second Error Prone PCR using as templatesrandom mutant-expressing transformants (JM109/pUMESDsy-H103L andpUMESDsy-H103L) obtained from the first Error Prone PCR using astemplates E. coli codon wild-type hydroxynitrile lyase-expressingtransformants (JM109/pUMESDsy and pUMESDsy) in Example 8.

FIG. 5 is a diagram showing the results of SDS-PAGE analysis on cellextract soluble fractions (S) and insoluble fractions (I) preparedrespectively from an E. coli codon wild-type hydroxynitrilelyase-expressing transformant (JM109/pUMESDsy), a plant codon wild-typehydroxynitrile lyase-expressing transformant (JM109/pUMESD), atransformant expressing an improved hydroxynitrile lyase obtained byintroducing H103L mutation into the E. coli codon wild-typehydroxynitrile lyase (JM109/pUMESDsy−H103L), and a transformantexpressing an improved hydroxynitrile lyase obtained by introducingH103L mutation into the plant codon wild-type hydroxynitrile lyase(pUMESD-H103L) in Example 9.

FIG. 6 (A) is a graph showing the hydroxynitrile lyase activities of E.coli codon wild-type hydroxynitrile lyase (His) and 9 types of mutantsobtained by substituting the histidine residue at position 103 of thewild-type amino acid sequence with other amino acids in Example 10. FIG.6 (B) is a graph showing the hydroxynitrile lyase activities of plantcodon wild-type hydroxynitrile lyase (His) and 10 types of mutantsobtained by substituting the histidine residue at position 103 of thewild-type amino acid sequence with other amino acids.

FIG. 7 is a diagram showing the results of SDS-PAGE analysis ofindividual fractions (T: total fraction; P: insoluble fraction; S:soluble fraction) of cell extracts obtained from flask cultivation at30° C. and 37° C. of a plant codon wild-type hydroxynitrilelyase-expressing transformant (JM109/pOXN103); V2I mutation-introduced,plant codon improved hydroxynitrile lyase-expressing transformant(JM109/pOXN103V2I); H103L mutation-introduced, plant codon improvedhydroxynitrile lyase-expressing transformant (JM109/pOXN103H103L); andV2I and H103L mutations-introduced, plant codon combined type improvedhydroxynitrile lyase-expressing transformant (JM109/pOXN103V2I+H103L) inExample 12.

FIG. 8 shows the results of cell concentration, activity (specificactivity and liquid activity) and SDS-PAGE (evaluation of T: totalfraction; P: insoluble fraction; S: soluble fraction) in the evaluationof jar cultivation of V2I mutation-introduced, plant codon improvedhydroxynitrile lyase-expressing transformant (C600/pOXN103V2I) and V2Iand H103L mutations-introduced, plant codon combined type improvedhydroxynitrile lyase-expressing transformant (C600/pOXN103V2I+H103L) inExample 12.

FIG. 9 shows the results of SDS-PAGE analysis in Example 13 whichexamined the effect of difference in the codon encoding H103 residue inthe E. coli codon wild-type hydroxynitrile lyase upon expression levels.

FIG. 10 shows the results of SDS-PAGE analysis on purified wild-typehydroxynitrile lyase (MeHNL) and purified H103M improved hydroxynitrilelyase (MeHNL-H103M) in Example 14.

FIG. 11 shows the results of SDS-PAGE analysis on 10 μg of proteinsamples derived from soluble fractions of lysine residue-substitutedmutants in Examples 15 and 16.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinbelow, embodiments of the present invention will be described.These embodiments are provided only to illustrate the present invention,and the present invention is not limited to these embodiments. Thepresent invention may be practiced in various ways without departingfrom the gist of the present invention. All publications, and patentpublications such as unexamined patent publications and issued patentpublications cited herein are incorporated herein by reference in theirentirety.

The present invention is based on a finding that it is possible togreatly improve hydroxynitrile lyase activity per transformant bysubstituting at least one amino acid residue in the amino acid sequenceof a wild-type hydroxynitrile lyase with another amino acid.

(I) Hydroxynitrile Lyase Activity

The term “hydroxynitrile lyase activity” used in the presentspecification means both the activity to catalyze reactions producing acyanohydrin from either ketone or aldehyde and a cyanide compound(hereinafter, referred to as “synthesis activity”) and the activity tocatalyze reverse reactions thereof (hereinafter, referred to as“degradation activity”). In the present invention, the synthesisactivity may be calculated by measuring the amount of (S)-mandelonitrilegenerated from benzaldehyde. Generation of mandelonitrile may bequantitatively determined, for example, by HPLC. The degradationactivity may be calculated by measuring the amount of benzaldehydegenerated from mandelonitrile (substrate). Generation of benzaldehydemay be quantitatively determined, for example, by tracing increase inabsorbance at 280 nm when an improved hydroxynitrile lyase and racemicmandelonitrile are added to a sodium citrate buffer.

The present invention relates to an improved hydroxynitrile lyaseobtainable by substituting some amino acid residues of the amino acidsequence of a wild-type hydroxynitrile lyase with other amino acids. Theimproved hydroxynitrile lyase of the present invention is, as describedlater, characterized by its hydroxynitrile lyase activity pertransformant being higher than the hydroxynitrile lyase activity pertransformant of the corresponding wild-type hydroxynitrile lyase. Theessential cause which brought about the above-described improvement inactivity per transformant may be any cause as long as it is attributableto the introduction of mutation. For example, improvement in thespecific activity per enzyme protein itself, improvement in the abilityto form active conformation, increase in the expression level intransformants (in particular, soluble fraction) and the like may beenumerated. Further, improvement in resistances, such as resistance tometal ions, resistance to organic solvents, thermal resistance, acidresistance or alkali resistance, may also be enumerated. Therefore, theimproved hydroxynitrile lyase of the present invention may be ahydroxynitrile lyase whose hydroxynitrile lyase activity pertransformant has been increased by the introduction of mutation. Theimproved hydroxynitrile lyase of the present invention also includes ahydroxynitrile lyase whose specific activity per enzyme protein itselfhas been increased; a hydroxynitrile lyase whose ability to form activeconformation has been increased; or a hydroxynitrile lyase whoseexpression level per transformant has been increased.

As used herein, the term “activity per transformant” meanshydroxynitrile lyase activity per transformant culture equipment, perculture broth, per amount of transformant (wet or dry), per (crude)enzyme solution, per soluble fraction, per weight of protein or the likein enzyme solution. High (low) in this activity means thathydroxynitrile lyase activity (specific activity, liquid activity) perunit weight of protein in enzyme solution or the like, or per unitvolume of enzyme solution or the like is higher (lower) than that ofcontrol.

As used herein, the term “specific activity” means hydroxynitrile lyaseactivity per unit weight of protein or per unit cell mass.

As used herein, the term “liquid activity” means hydroxynitrile lyaseactivity per unit volume of solution.

(II) Hydroxynitrile Lyase

(II-1) Wild-Type Hydroxynitrile Lyase

The improved hydroxynitrile lyase of the present invention has beenimproved by introducing a mutation into a wild-type hydroxynitrilelyase. The source of this wild-type hydroxynitrile lyase is notparticularly limited. However, plant-derived hydroxynitrile lyases arepreferable. As used herein, the term “wild-type hydroxynitrile lyase”refers to a hydroxynitrile lyase which can be isolated from an organism(such as plant) in the natural world and means that this hydroxynitrilelyase has no intentional or unintentional alterations in the sequence ofamino acids constituting the enzyme (such as deletion or insertion ofamino acids or substitution with other amino acids) and that thishydroxynitrile lyase is retaining its properties derived from nature. Inthe present invention, the wild-type hydroxynitrile lyase may be(S)-hydroxynitrile lyase or (R)-hydroxynitrile lyase. Preferably,(S)-hydroxynitrile lyase is used. Examples of plants from which thewild-type hydroxynitrile lyase is derived include, but are not limitedto, cassava (Manihot esculenta), Para rubber tree (Hevea brasiliensis),sorghum (Sorghum bicolor), almond (Prunus amygdalus) and tallow wood(Ximenia americana). Among them, cassava or Para rubber tree ispreferable. For example, the amino acid sequence of cassava-derivedwild-type hydroxynitrile lyase is disclosed in GenBank/EMBL accessionnumber Z29091 and shown in SEQ ID NO: 1. The amino acid sequence of Pararubber tree-derived wild-type hydroxynitrile lyase is disclosed inGenBank/EMBL accession number U40402 and shown in SEQ ID NO: 102.

In the present specification, the cassava-derived wild-typehydroxynitrile lyase is mainly taken as an example to describe thepresent invention. However, as described above, the source ofhydroxynitrile lyase is not particularly limited. Even when a wild-typehydroxynitrile lyase other than the one derived from cassava is used, itis possible to improve hydroxynitrile lyase activity per transformant byapplying the site of mutation or the type of amino acid or nucleotidesequence to be mutated disclosed in the present invention. Somewild-type hydroxynitrile lyases have high homology in amino acidsequence though they are derived from different organism species. Forexample, cassava-derived wild-type hydroxynitrile lyase and Para rubbertree-derived wild-type hydroxynitrile lyase may be enumerated. They have74% amino acid sequence homology (Japanese Unexamined Patent PublicationNo. 11-508775). In the present invention, high homology refers to 60% ormore homology, preferably 75% or more homology, and particularlypreferably 90% or more homology. Even when homology over the full lengthof amino acid sequence is low, some hydroxynitrile lyases have highsimilarity in the secondary structure (e.g., the structure or positionof α helix or β sheet), tertiary structure or quaternary structure ofprotein. It is preferable that the wild-type hydroxynitrile lyase usedin the present invention derived from an organism other than cassava orPara rubber tree have the above-described homology and similarity.

(II-2) Improved Hydroxynitrile Lyase

The “improved hydroxynitrile lyase” in the present invention is definedas a hydroxynitrile lyase characterized by having a mutation ofsubstitution of at least one amino acid residue in the amino acidsequence of a corresponding wild-type hydroxynitrile lyase with anotheramino acid mainly using DNA recombination techniques and by itshydroxynitrile lyase activity per transformant being higher than that ofthe wild-type hydroxynitrile lyase. The “improved hydroxynitrile lyase”is included in the scope of the present invention.

In the present invention, preferably, the “improved hydroxynitrilelyase” has any one of the characteristics described in the following (A)to (G), and yet its hydroxynitrile lyase activity per transformant ishigher than the hydroxynitrile lyase activity per transformant intowhich the corresponding wild-type hydroxynitrile lyase gene isintroduced.

(A) an improved hydroxynitrile lyase in which the amino acid residue atposition 2 of the amino acid sequence of a wild-type hydroxynitrilelyase is substituted with another amino acid residue;

(B) an improved hydroxynitrile lyase in which the histidine residue atposition 103 or a neighboring position thereto of the amino acidsequence of a wild-type hydroxynitrile lyase is substituted with anotheramino acid residue;

(C) an improved hydroxynitrile lyase in which at least one lysineresidue of the amino acid sequence of a wild-type hydroxynitrile lyaseis substituted with another amino acid residue;

(D) an improved hydroxynitrile lyase in which the amino acid residue atposition 2 and the histidine residue at position 103 or a neighboringposition thereto of the amino acid sequence of a wild-typehydroxynitrile lyase are substituted with other amino acid residues;

(E) an improved hydroxynitrile lyase in which the amino acid residue atposition 2 and at least one lysine residue of the amino acid sequence ofa wild-type hydroxynitrile lyase are substituted with other amino acidresidues;

(F) an improved hydroxynitrile lyase in which the histidine residue atposition 103 or a neighboring position thereto and at least one lysineresidue of the amino acid sequence of a wild-type hydroxynitrile lyaseare substituted with other amino acid residues;

(G) an improved hydroxynitrile lyase in which the amino acid residue atposition 2, the histidine residue at position 103 or a neighboringposition thereto and at least one lysine residue of the amino acidsequence of a wild-type hydroxynitrile lyase are substituted with otheramino acid residues.

In (A), (D), (E), or (G) above, the substituent amino acid whichreplaces the amino acid residue at position 2 (e.g., valine) of awild-type hydroxynitrile lyase is not particularly limited. Any aminoacid other than the one at position 2 of the wild-type hydroxynitrilelyase may be used as long as the activity of the resultant polypeptideafter the substitution per transformant becomes higher than the activityof the wild-type hydroxynitrile lyase per transformant. The substituentamino acid which replaces the amino acid residue at position 2 isselected from 19 types of amino acids excluding valine incassava-derived hydroxynitrile lyase; and selected from 19 types ofamino acids excluding alanine in Para rubber tree-derived hydroxynitrilelyase. The substituent amino acid which replaces the amino acid residueat position 2 is preferably lysine, asparagine, isoleucine, arginine,glutamine, proline, threonine, tyrosine, leucine, methionine, serine,glutamic acid, alanine, glycine or aspartic acid; more preferablylysine, asparagine, isoleucine, arginine, glutamine, proline, threonine,tyrosine, leucine, methionine, serine or glutamic acid; still morepreferably lysine, asparagine, isoleucine, arginine, glutamine, proline,threonine, tyrosine, leucine, methionine or serine; and particularlypreferably lysine, asparagine, isoleucine, arginine or glutamine.

As a finding relating to the stability of proteins in cells,relationship between the amino acid at position 2 and formylmethionineprocessing or the N-end rule has been reported. The “relationshipbetween the amino acid at position 2 and formylmethionine processing”means that whether or not a protein is ready to undergo processing bymethionine aminopeptidase is decided by the type of the amino acid atposition 2 of the protein and that the larger the side chains of theamino acid at position 2 are, the more difficult it is for the proteinto undergo the processing. The “N-end rule” says that when theN-terminus residue of a protein is arginine, lysine, leucine,phenylalanine, tyrosine, tryptophan or the like, the protein in cells islow in stability and degraded rapidly. According to these relationshipor rule, it was considered that there is no significant difference inthe stability of proteins when comparing, for example, a protein whichhas valine at position 2 of SEQ ID NO: 1 and a protein which hasisoleucine at position 2 of SEQ ID NO: 1. However, one of thecharacteristics of the present invention resides in a point that theexpression level of a hydroxynitrile lyase is improved by substitutingthe amino acid at position 2 with another amino acid as described aboveand, as a result, hydroxynitrile lyase activity per transformant isimproved. Therefore, the present invention can not be explained by theexisting, above-described relationship or rule. It is believed that thepresent invention results from a thoroughly new principle.

In (B), (D), (F), or (G) above, the substituent amino acid whichreplaces the histidine residue at position 103 of a wild-typehydroxynitrile lyase is not particularly limited. Any amino acid otherthan histidine may be used as long as the activity of the resultantpolypeptide after the substitution per transformant becomes higher thanthe activity of the wild-type hydroxynitrile lyase per transformant. Thesubstituent amino acid which replaces the histidine residue at position103 is preferably an amino acid having one or both of the followingproperties (a) and (b):

(a) an amino acid containing one or two nitrogen atoms in its molecule;

(b) a neutral amino acid.

Here, the “amino acid containing one or two nitrogen atoms in itsmolecule” in (a) refers to, for example, alanine, asparagine, asparticacid, cysteine, glutamine, glutamic acid, glycine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine or valine. The “neutral amino acid” in (b) refersto an amino acid other than acidic amino acids and basic amino acids.Specific examples of neutral amino acids include methionine, leucine,isoleucine, valine, cysteine, glutamine, serine, threonine, alanine,tryptophan, phenylalanine, asparagine, tyrosine, glycine and proline(“Dictionary of Biochemistry” published by Tokyo Kagaku Dojin). Thesubstituent amino acid which replaces the histidine residue at position103 is not particularly limited; any amino acid having the aboveproperty (a) and/or property (b) may be used. Preferably, methionine,leucine, isoleucine, valine, cysteine, glutamine, serine, threonine,alanine or tryptophan is used. More preferably, methionine, leucine,isoleucine, valine, cysteine or tryptophan is used.

Further, an improved hydroxynitrile lyase in which the histidine residueat a neighboring position to position 103 of the amino acid sequence ofa wild-type hydroxynitrile lyase is substituted with another amino acidis also included in the present invention. In wild-type hydroxynitrilelyases not derived from cassava or Para rubber tree, a histidine residuecorresponding to the histidine residue at position 103 of cassava orPara rubber tree hydroxynitrile lyase may exist at a neighboringposition to position 103. In such a case, the histidine residue existingat a neighboring position to position 103 may be substituted withanother amino acid in the same manner as described above.

In the present invention, a histidine residue “at a neighboring positionto position 103” means a histidine residue located at a position from 93to 113, preferably from 98 to 108, and more preferably from 100 to 106.

The position of a histidine residue corresponding to the histidineresidue at position 103 of cassava or Para rubber tree hydroxynitrilelyase may be ascertained by, for example, aligning the amino acidsequence of cassava- or Para rubber tree-derived wild-typehydroxynitrile lyase and the amino acid sequence of a hydroxynitrilelyase of interest. Alignment of amino acid sequences may be performedusing, for example, ClustalW available from the website of DNA Data Bankof Japan (http://www.ddbj.nig.ac.jp/search/clustalw-j.html).

In (C), (E), (F) or (G) above, the lysine residue to be substituted withanother amino acid residue is not particularly limited. Any lysineresidue present in the amino acid sequence of the wild-typehydroxynitrile lyase may be selected as long as the activity of theresultant polypeptide after the substitution per transformant becomeshigher than the activity of the wild-type hydroxynitrile lyase pertransformant. The lysine residue to be substituted with another aminoacid residue is preferably at least one lysine residue present in aregion from positions 175 to 224 in the amino acid sequence of awild-type hydroxynitrile lyase. More preferably, one or more residuesselected from the lysine residues at positions 176, 199 and 224 incassava-derived wild-type hydroxynitrile lyase and one or more residuesselected from the lysine residues at positions 175, 198 and 223 in Pararubber tree-derived wild-type hydroxynitrile lyase are substituted,respectively.

The substituent amino acids which replace these lysine residues arepreferably amino acids having one or both of the following properties(a) and (b):

(a) an amino acid containing one or two nitrogen atoms in its molecule;

(b) a neutral amino acid.

Here, the “amino acid containing one or two nitrogen atoms in itsmolecule” in (a) is as defined above. The “neutral amino acid” in (b) isas defined above. The most preferable substituent amino acid whichreplaces these lysine residues is proline.

As preferred embodiments of the improved hydroxynitrile lyases of (A) to(C) above, specifically, the following hydroxynitrile lyases may beenumerated, for example.

(A) An improved hydroxynitrile lyase in which the valine or alanineresidue at position 2 of the amino acid sequence of a wild-typehydroxynitrile lyase as shown in SEQ ID NO: 1 or SEQ ID NO: 102 issubstituted with any amino acid selected from the group consisting oflysine, asparagine, isoleucine, arginine, glutamine, proline, threonine,tyrosine, leucine, methionine, serine and glutamic acid.(B) An improved hydroxynitrile lyase in which the histidine residue atposition 103 of the amino acid sequence of a wild-type hydroxynitrilelyase as shown in SEQ ID NO: 1 or SEQ ID NO: 102 is substituted with anyamino acid selected from the group consisting of methionine, leucine,isoleucine, valine, cysteine, glutamine, serine, threonine, alanine andtryptophan.(C) An improved hydroxynitrile lyase in which one of the lysine residuesat positions 176, 199 and 224, the lysine residues at positions 176 and199, the lysine residues at positions 176 and 224, the lysine residuesat positions 199 and 224, or the lysine residues at positions 176, 199and 224 in the amino acid sequence of a wild-type hydroxynitrile lyaseas shown in SEQ ID NO: 1 are substituted with proline residues; and animproved hydroxynitrile lyase in which one of the lysine residues atpositions 175, 198 and 223, the lysine residues at positions 175 and198, the lysine residues at positions 175 and 223, the lysine residuesat positions 198 and 223, or the lysine residues at positions 175, 198and 223 in the amino acid sequence of a wild-type hydroxynitrile lyaseas shown in SEQ ID NO: 102 are substituted with proline residues.

Further, the improved hydroxynitrile lyases of (D) to (G) above areimproved hydroxynitrile lyases provided with two or three of theembodiments described in (A) to (C) above. For example, (D) is acombination of (A) and (B), and represents an improved hydroxynitrilelyase in which the valine or alanine residue at position 2 of the aminoacid sequence of a wild-type hydroxynitrile lyase as shown in SEQ ID NO:1 or SEQ ID NO: 102 is substituted with any amino acid selected from thegroup consisting of lysine, asparagine, isoleucine, arginine, glutamine,proline, threonine, tyrosine, leucine, methionine, serine and glutamicacid, and yet the histidine residue at position 103 thereof issubstituted with any amino acid selected from the group consisting ofmethionine, leucine, isoleucine, valine, cysteine, glutamine, serine,threonine, alanine and tryptophan.

It should be noted here that SEQ ID NO: 1 shows the amino acid sequenceof cassava-derived wild-type hydroxynitrile lyase, and SEQ ID NO: 102shows the amino acid sequence of Para rubber tree-derived wild-typehydroxynitrile lyase.

The present invention also includes an improved hydroxynitrile lyase inwhich the phenylalanine residue at position 125 in SEQ ID NO: 1 or SEQID NO: 102 is substituted with leucine; an improved hydroxynitrile lyasein which the threonine residue at position 205 therein is substitutedwith serine; or an improved hydroxynitrile lyase in which the asparagineresidue at position 235 therein is substituted with glycine.

Further, the improved hydroxynitrile lyase of the present inventionincludes within its scope a polypeptide which has a characteristic asdescribed in (A) to (G) above (i.e., retaining the embodiment ofsubstitution), which consists of an amino acid sequence having deletion,substitution or addition of one or several (e.g., about 1-10, preferably1-5) amino acids other than those located at the substitution positionsspecified in (A) to (G), and whose hydroxynitrile lyase activity pertransformant is higher than that of the wild-type hydroxynitrile lyase.Examples of such polypeptides include the following embodiments.

(A) A polypeptide which consists of an amino acid sequence of awild-type hydroxynitrile lyase wherein the amino acid residue atposition 2 is substituted with another amino acid residue, the aminoacid sequence further having deletion, substitution or addition of oneor several amino acids other than the substituent amino acid at position2; and yet which has hydroxynitrile lyase activity.(B) A polypeptide which consists of an amino acid sequence of awild-type hydroxynitrile lyase wherein the histidine residue at position103 or a neighboring position thereto is substituted with another aminoacid residue, the amino acid sequence further having deletion,substitution or addition of one or several amino acids other than thesubstituent amino acid at position 103 or a neighboring positionthereto; and yet which has hydroxynitrile lyase activity.(C) A polypeptide which consists of an amino acid sequence of awild-type hydroxynitrile lyase wherein at least one lysine residue issubstituted with another amino acid residue, the amino acid sequencefurther having deletion, substitution or addition of one or severalamino acids other than the substituent amino acid for the lysineresidue; and yet which has hydroxynitrile lyase activity.(D) A polypeptide which consists of an amino acid sequence of awild-type hydroxynitrile lyase wherein the amino acid residue atposition 2 and the histidine residue at position 103 or a neighboringposition thereto are substituted with other amino acid residues, theamino acid sequence further having deletion, substitution or addition ofone or several amino acids other than the substituent amino acids atposition 2 and position 103 or a neighboring position thereto; and yetwhich has hydroxynitrile lyase activity.(E) A polypeptide which consists of an amino acid sequence of awild-type hydroxynitrile lyase wherein the amino acid residue atposition 2 and at least one lysine residue are substituted with otheramino acid residues, the amino acid sequence further having deletion,substitution or addition of one or several amino acids other than thesubstituent amino acid at position 2 and the substituent amino acid forthe lysine residue; and yet which has hydroxynitrile lyase activity.(F) A polypeptide which consists of an amino acid sequence of awild-type hydroxynitrile lyase wherein the histidine residue at position103 or a neighboring position thereto and at least one lysine residueare substituted with other amino acid residues, the amino acid sequencefurther having deletion, substitution or addition of one or severalamino acids other than the substituent amino acid at position 103 or aneighboring position thereto and the substituent amino acid for thelysine residue; and yet which has hydroxynitrile lyase activity.(G) A polypeptide which consists of an amino acid sequence of awild-type hydroxynitrile lyase wherein the amino acid residue atposition 2, the histidine residue at position 103 or a neighboringposition thereto and at least one lysine residue are substituted withother amino acid residues, the amino acid sequence further havingdeletion, substitution or addition of one or several amino acids otherthan the substituent amino acid at position 2, the substituent aminoacid at position 103 or a neighboring position thereto and thesubstituent amino acid for the lysine residue; and yet which hashydroxynitrile lyase activity.

It should be noted that amino acids are sometimes expressed in thecommonly used three-letter or one-letter abbreviation codes in thepresent specification. An alphabet placed before a numerical figure mayrepresent the one-letter abbreviation of the amino acid beforesubstitution, and an alphabet placed after the numerical figure mayrepresent the one-letter abbreviation of the amino acid aftersubstitution. For example, when lysine at position 176 is substitutedwith proline, an expression “K176P” may be used. This method ofexpression is applicable to other substitutions.

(III) Hydroxynitrile Lyase Gene

(III-1) Wild-Type Hydroxynitrile Lyase Gene

Genetic sequences of some wild-type hydroxynitrile lyases have beenelucidated. For example, the genetic sequence for the above-describedcassava-derived wild-type hydroxynitrile lyase is shown in SEQ ID NO: 2(GenBank; accession number Z29091). In the present invention, this issometimes referred to as the “plant codon wild-type hydroxynitrile lyasegene”. Further, the genetic sequence for the above-described Para rubbertree-derived wild-type hydroxynitrile lyase is shown in SEQ ID NO: 103(GenBank; accession number U40402).

As one example of method for obtaining the plant codon wild-typehydroxynitrile lyase gene, a method may be given which comprisesextracting total RNA or mRNA containing the mRNA of the gene from aplant and synthesizing cDNA by conventional methods (see, for example,Molecular Cloning, A Laboratory Manual 2nd ed. (Cold Spring Harbor Press(1989)). Briefly, primers are designed based on known information aboutthe genetic sequence of the plant codon wild-type hydroxynitrile lyase.Then, the gene encoding hydroxynitrile lyase is amplified by PCR usingthe primers to thereby obtain the plant codon wild-type hydroxynitrilelyase gene. Alternatively, it is also possible to chemically synthesizethe full length of the plant codon wild-type hydroxynitrile lyase geneusing, for example, PCR with a combination of synthetic oligo-DNAs(assembly PCR) based on known information about the genetic sequence.For example, the plant codon wild-type hydroxynitrile lyase gene isdivided into several regions (each consisting of about 50 bp), and anumber of oligo-nucleotides having an overlap (e.g., about 20 bp) withthe adjacent region at both ends are designed and synthesized. Then,these oligo-nucleotides are annealed with each other by PCR to therebyamplify the plant codon wild-type hydroxynitrile lyase gene.

Recently, it has become possible to produce a protein of interest in aheterologous host using recombinant DNA techniques. It is known that useof those codons frequently used in the host organism improves theexpression level of the protein in many cases. As used herein, frequencyin the use of codons means the frequency at which codons are used in theprocess of information conversion from nucleotide sequence to amino acidsequence, and codon means the positioning of three nucleotides in mRNA.In the above-mentioned process of information conversion, the threenucleotides as a unit are translated into one amino acid. Since 64 typesof codons correspond to 20 types of amino acids, there is degeneracy inthe genetic code and one amino acid has 1 to 6 types of codons whichspecify the amino acid. For example, valine has 4 codons of GUU, GUC,GUA and GUG. When there are a plurality of codons for one amino acid,the organism does not use those codons equally but uses specific codonsin a biased manner at ratios characteristic to each organism. Suchfrequency in the use of codons (codon usage) by each organism ispartially stored in databases and can be available from Codon UsageDatabase (http://www.kazusa.or.jp/codon/).

As used herein, the term “high” in frequency means higher than thelowest frequency when there are a plurality of codons specifying oneamino acid, and may not mean the highest frequency. When there is onlyone codon for one amino acid (e.g., methionine and tryptophan), thecodon is used regardless of frequency. However, considering expressionefficiency in hosts, it is preferable to use high frequency codons inhigh expression genes or the highest frequency codons in the host. Morespecifically, when E. coli K12 strain is used as a host, the highestfrequency codons (Table 2) may be known from the table of codon usageshown in Table 1. Therefore, when E. coli K12 strain is used as a host,codons of a gene of interest to be expressed may be converted to thosecodons shown in Table 2 using genetic engineering techniques. Forexample, when the codon used for valine is GTA in the plant codonwild-type hydroxynitrile lyase gene obtained by extracting total RNA ormRNA comprising the mRNA of the gene from cassava and synthesizing cDNAtherefrom, as described above, GTA may be converted to GTG which is thehighest frequency codon for valine in E. coli K12 strain.

TABLE 1 Codon Usage in E. coli K12 Strain Escherichia coli K12 [gbbct]:5089 CDS's (1608122 codons) fields: [triplet] [frequency: per thousand]([number]) 2nd Nucleotide 1st Nucleotide U C A G U Phe UUU 22.4 (35982)Ser UCU 8.5 (13687) Tyr UAU 16.3 (26266) Cys UGU 5.2 (8340) UUC 16.6(26678) UCC 8.6 (13849) UAC 12.3 (19728) UGC 6.4 (10347) C Leu UUA 13.9(22376) UCA 7.2 (11511) termination UAA 2.0 (3246) termination UGA 0.9(1468) UUG 13.7 (22070) UCG 8.9 (14379) termination UAG 0.2 (378) TrpUGG 15.3 (24615) CUU 11.0 (17754) Pro CCU 7.1 (11340) His CAU 12.9(20728) Arg CGU 21.0 (33694) CUC 11.0 (17723) CCC 5.5 (8915) CAC 9.7(15595) CGC 22.0 (35306) CUA 3.9 (6212) CCA 8.5 (13707) Gln CAA 15.4(24835) CGA 3.6 (5716) CUG 52.7 (84673) CCG 23.2 (37328) CAG 28.8(46319) CGG 5.4 (8684) A Ile AUU 30.4 (48818) Thr ACU 9.0 (14397) AsnAAU 17.7 (28465) Ser AGU 8.8 (14092) AUC 25.0 (40176) ACC 23.4 (37624)AAC 21.7 (34912) AGC 16.1 (25843) AUA 4.3 (6962) ACA 7.1 (11366) Lys AAA33.6 (54097) Arg AGA 2.1 (3337) Met AUG 27.7 (44614) ACG 14.4 (23124)AAG 10.2 (16401) AGG 1.2 (1987) (Initiation) G Val GUU 18.4 (29569) AlaGCU 15.4 (24719) Asp GAU 32.2 (51852) Gly GGU 24.9 (40019) GUC 15.2(24477) GCC 25.5 (40993) GAC 19.0 (30627) GGC 29.4 (47309) GUA 10.9(17508) GCA 20.3 (32666) Glu GAA 39.5 (63517) GGA 7.9 (12776) GUG 26.2(42212) GCG 33.6 (53988) GAG 17.7 (28522) GGG 11.0 (17704) Coding GC51.80% 1st letter GC 58.89% 2nd letter GC 40.72% 3rd letter GC 55.79%Codons expressed in bold letters are frequently used codons in E. coli.This Table is based onhttp://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=Eschericia+coli+K12+[gbbct]as of Feb. 20, 2004.

TABLE 2 Highest Frequency Codons in E. coli K12 Strain Amino Acid AminoAcid (three-letter abbreviation) codon (three-letter abbreviation) codonAla GCG Thr ACC Val GUG Cys UGC Leu CUG Gln CAG Ile AUU Asn AAC Pro CCGTyr UAU Phe UUU Lys AAA Trp UGG Arg CGC Met AUG His CAU Gly GGC Asp GAUSer AGC Glu GAA

The region in which codons are converted may be any region within thecoding sequence (CDS). Every codon or a part thereof (at one or severalsites) in the CDS may be converted. In the case of the cassava-derivedwild-type hydroxynitrile lyase in the present invention, the number ofamino acid residues in which codons are converted may be one or more,preferably 1 to 100, more preferably 10 to 70 in the 258 amino acidresidues.

A genetic sequence thus composed of codons suitable for the host andencoding the amino acid sequence of a wild-type hydroxynitrile lyase isdesignated a “host codon wild-type hydroxynitrile lyase gene” in thepresent invention. When the host is E. coli, the gene is sometimescalled the “E. coli codon wild-type hydroxynitrile lyase gene” anddiscriminated from the above-described “plant codon wild-typehydroxynitrile lyase gene”. When the term “hydroxynitrile lyase gene” isused without the expression of “XXX codon” at the beginning, it isintended that this gene is not limited to plant codon gene or E. colicodon gene or it encompasses the both genes.

When the number of codons to be converted is relatively small, the hostcodon hydroxynitrile lyase gene may be prepared from the plant codonwild-type hydroxynitrile lyase gene using the site-directed mutagenesismethod described, for example, in Molecular Cloning, A Laboratory Manual2nd ed., Cold Spring Harbor Laboratory Press (1989) and CurrentProtocols in Molecular Biology, John Wiley & Sons (1987-1997). Recently,it has become possible to perform site-directed mutagenesis relativelyeasily using mutagenesis introduction kits such as QuickChange™Site-Directed Mutagenesis Kit (Stratagene), GeneTailor™ Site-DirectedMutagenesis System (Invitrogen) or TaKaRa Site-Directed MutagenesisSystem (e.g., Mutan-K, Mutan-Super Express Km; Takara Bio).Alternatively, it is also possible to synthesize the full length of hostcodon wild-type hydroxynitrile lyase gene in which a large number ofcodons are converted to highest frequency codons in the relevant host,by PCR using a combination of synthetic oligo-DNAs (assembly PCR) asdescribed earlier. As an example of E. coli codon wild-typehydroxynitrile lyase gene, the E. coli codon wild-type hydroxynitrilelyase gene consisting of the nucleotide sequence as shown in SEQ ID NO:3 described later in Examples may be given.

(III-2) Improved Hydroxynitrile Lyase Gene

The improved hydroxynitrile lyase gene in the present invention means agene encoding the improved hydroxynitrile lyase enzyme protein describedin (II-2) above. The improved hydroxynitrile lyase gene of the presentinvention include, for example, genes encoding improved hydroxynitrilelyases which have such amino acid substitution mutations as described in(A) to (G) in (II-2) above in the wild-type hydroxynitrile lyaserepresented by the amino acid sequence of SEQ ID NO: 1 (derived formcassava) or SEQ ID NO: 102 (derived from Para rubber tree). The codonsof the wild-type hydroxynitrile lyase which are bases upon which theimproved hydroxynitrile lyase gene is prepared may be either plantcodons or host codons. Examples of the improved hydroxynitrile lyasegene of the present invention include the following embodiments.

(A)

The sequence as shown in SEQ ID NO: 2 wherein the nucleotides GTA atpositions 4 to 6 are substituted with AAA or AAG, preferably thenucleotides G and T at positions 4 and 5 are substituted with A and A,respectively (this sequence encodes an improved hydroxynitrile lyasewherein the valine residue at position 2 is substituted with lysine);

The sequence as shown in SEQ ID NO: 2 wherein the nucleotides GTA atpositions 4 to 6 are substituted with AAC or AAT, preferably thenucleotides GTA at positions 4 to 6 are substituted with AAC (thissequence encodes an improved hydroxynitrile lyase wherein the valineresidue at position 2 is substituted with asparagine);

The sequence as shown in SEQ ID NO: 2 wherein the nucleotides GTA atpositions 4 to 6 are substituted with ATA, ATC or ATT, preferably thenucleotides G and A at positions 4 and 6 are substituted with A and C,respectively (this sequence encodes an improved hydroxynitrile lyasewherein the valine residue at position 2 is substituted withisoleucine);

The sequence as shown in SEQ ID NO: 2 wherein the nucleotides GTA atpositions 4 to 6 are substituted with AGA, AGQ CGA, CGC, CGG or CGT,preferably the nucleotides GTA at positions 4 to 6 are substituted withCGT (this sequence encodes an improved hydroxynitrile lyase wherein thevaline residue at position 2 is substituted with arginine);

The sequence as shown in SEQ ID NO: 2 wherein the nucleotides GTA atpositions 4 to 6 are substituted with CAA or CAG, preferably thenucleotides GTA at positions 4 to 6 are substituted with CAG (thissequence encodes an improved hydroxynitrile lyase wherein the valineresidue at position 2 is substituted with glutamine);

The sequence as shown in SEQ ID NO: 2 wherein the nucleotides GTA atpositions 4 to 6 are substituted with CCA, CCC, CCG or CCT, preferablythe nucleotides GTA at positions 4 to 6 are substituted with CCG (thissequence encodes an improved hydroxynitrile lyase wherein the valineresidue at position 2 is substituted with proline);

The sequence as shown in SEQ ID NO: 2 wherein the nucleotides GTA atpositions 4 to 6 are substituted with ACA, ACC, ACG or ACT, preferablythe nucleotides GTA at positions 4 to 6 are substituted with ACC (thissequence encodes an improved hydroxynitrile lyase wherein the valineresidue at position 2 is substituted with threonine);

The sequence as shown in SEQ ID NO: 2 wherein the nucleotides GTA atpositions 4 to 6 are substituted with TAC or TAT, preferably thenucleotides GTA at positions 4 to 6 are substituted with TAC (thissequence encodes an improved hydroxynitrile lyase wherein the valineresidue at position 2 is substituted with tyrosine);

The sequence as shown in SEQ ID NO: 2 wherein the nucleotides GTA atpositions 4 to 6 are substituted with TTA, TTG, CTA, CTC, CTG or CTT,preferably the nucleotides G and A at positions 4 and 6 are substitutedwith C and G, respectively (this sequence encodes an improvedhydroxynitrile lyase wherein the valine residue at position 2 issubstituted with leucine);

The sequence as shown in SEQ ID NO: 2 wherein the nucleotides GTA atpositions 4 to 6 are substituted with ATG, i.e., the nucleotides G and Aat positions 4 and 6 are substituted with A and G, respectively (thissequence encodes an improved hydroxynitrile lyase wherein the valineresidue at position 2 is substituted with methionine);

The sequence as shown in SEQ ID NO: 2 wherein the nucleotides GTA atpositions 4 to 6 are substituted with AGC, AGT, TCA, TCC, TCG or TCT,preferably the nucleotides GTA at positions 4 to 6 are substituted withAGC (this sequence encodes an improved hydroxynitrile lyase wherein thevaline residue at position 2 is substituted with serine);

The sequence as shown in SEQ ID NO: 2 wherein the nucleotides GTA atpositions 4 to 6 are substituted with GAA or GAQ preferably thenucleotide T at position 5 is substituted with A (this sequence encodesan improved hydroxynitrile lyase wherein the valine residue at position2 is substituted with glutamic acid);

The sequence as shown in SEQ ID NO: 2 wherein the nucleotides GTA atpositions 4 to 6 are substituted with GCA, GCC, GCG or GCT, preferablythe nucleotides T and A at positions 5 and 6 are substituted with C andT, respectively (this sequence encodes an improved hydroxynitrile lyasewherein the valine residue at position 2 is substituted with alanine);

The sequence as shown in SEQ ID NO: 2 wherein the nucleotides GTA atpositions 4 to 6 are substituted with GGA, GGC, GGG or GGT, preferablythe nucleotides T and A at positions 5 and 6 are substituted with G andC, respectively (this sequence encodes an improved hydroxynitrile lyasewherein the valine residue at position 2 is substituted with glycine);

The sequence as shown in SEQ ID NO: 2 wherein the nucleotides GTA atpositions 4 to 6 are substituted with GAC or GAT, preferably thenucleotides T and A at positions 5 and 6 are substituted with A and C,respectively (this sequence encodes an improved hydroxynitrile lyasewherein the valine residue at position 2 is substituted with asparticacid);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides GTG atpositions 4 to 6 are substituted with AAA or AAG, preferably thenucleotides GTG at positions 4 to 6 are substituted with AAA (thissequence encodes an improved hydroxynitrile lyase wherein the valineresidue at position 2 is substituted with lysine);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides GTG atpositions 4 to 6 are substituted with AAC or AAT, preferably thenucleotides GTG at positions 4 to 6 are substituted with AAC (thissequence encodes an improved hydroxynitrile lyase wherein the valineresidue at position 2 is substituted with asparagine);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides GTG atpositions 4 to 6 are substituted with ATA, ATC or ATT, preferably thenucleotides G and G at positions 4 and 6 are substituted with A and C,respectively (this sequence encodes an improved hydroxynitrile lyasewherein the valine residue at position 2 is substituted withisoleucine);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides GTG atpositions 4 to 6 are substituted with AGA, AGG, CGA, CGC, CGG or CGT,preferably the nucleotides GTG at positions 4 to 6 are substituted withCGT (this sequence encodes an improved hydroxynitrile lyase wherein thevaline residue at position 2 is substituted with arginine);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides GTG atpositions 4 to 6 are substituted with CAA or CAG, preferably thenucleotides G and T at positions 4 and 5 are substituted with C and A,respectively (this sequence encodes an improved hydroxynitrile lyasewherein the valine residue at position 2 is substituted with glutamine);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides GTG atpositions 4 to 6 are substituted with CCA, CCC, CCG or CCT, preferablythe nucleotides G and T at positions 4 and 5 are substituted with C andC, respectively (this sequence encodes an improved hydroxynitrile lyasewherein the valine residue at position 2 is substituted with proline);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides GTG atpositions 4 to 6 are substituted with ACA, ACC, ACG or ACT, preferablythe nucleotides GTG at positions 4 to 6 are substituted with ACC (thissequence encodes an improved hydroxynitrile lyase wherein the valineresidue at position 2 is substituted with threonine);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides GTG atpositions 4 to 6 are substituted with TAC or TAT, preferably thenucleotides GTG at positions 4 to 6 are substituted with TAC (thissequence encodes an improved hydroxynitrile lyase wherein the valineresidue at position 2 is substituted with tyrosine);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides GTG atpositions 4 to 6 are substituted with TTA, TTQ CTA, CTC, CTG or CTT,preferably the nucleotide G at position 4 is substituted with C (thissequence encodes an improved hydroxynitrile lyase wherein the valineresidue at position 2 is substituted with leucine);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides GTG atpositions 4 to 6 are substituted with ATG, i.e., the nucleotide G atposition 4 is substituted with A (this sequence encodes an improvedhydroxynitrile lyase wherein the valine residue at position 2 issubstituted with methionine);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides GTG atpositions 4 to 6 are substituted with AGC, AGT, TCA, TCC, TCG or TCT,preferably the nucleotides GTG at positions 4 to 6 are substituted withAGC (this sequence encodes an improved hydroxynitrile lyase wherein thevaline residue at position 2 is substituted with serine);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides GTG atpositions 4 to 6 are substituted with GAA or GAQ preferably thenucleotides T and G at positions 5 and 6 are substituted with A and A,respectively (this sequence encodes an improved hydroxynitrile lyasewherein the valine residue at position 2 is substituted with glutamicacid);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides GTG atpositions 4 to 6 are substituted with GCA, GCC, GCG or GCT, preferablythe nucleotides T and G at positions 5 and 6 are substituted with C andT, respectively (this sequence encodes an improved hydroxynitrile lyasewherein the valine residue at position 2 is substituted with alanine);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides GTG atpositions 4 to 6 are substituted with GGA, GGC, GGG or GGT, preferablythe nucleotides T and G at positions 5 and 6 are substituted with G andC, respectively (this sequence encodes an improved hydroxynitrile lyasewherein the valine residue at position 2 is substituted with glycine);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides GTG atpositions 4 to 6 are substituted with GAC or GAT, preferably thenucleotides T and G at positions 5 and 6 are substituted with A and C,respectively (this sequence encodes an improved hydroxynitrile lyasewherein the valine residue at position 2 is substituted with asparticacid);

The sequence as shown in SEQ ID NO: 103 wherein the nucleotides GCA atpositions 4 to 6 are substituted with AAA or AAG, preferably thenucleotides G and C at positions 4 and 5 are substituted with A and A,respectively (this sequence encodes an improved hydroxynitrile lyasewherein the alanine residue at position 2 is substituted with lysine);

The sequence as shown in SEQ ID NO: 103 wherein the nucleotides GCA atpositions 4 to 6 are substituted with AAC or AAT, preferably thenucleotides GCA at positions 4 to 6 are substituted with AAC (thissequence encodes an improved hydroxynitrile lyase wherein the alanineresidue at position 2 is substituted with asparagine);

The sequence as shown in SEQ ID NO: 103 wherein the nucleotides GCA atpositions 4 to 6 are substituted with ATA, ATC or ATT, preferably thenucleotides GCA at positions 4 to 6 are substituted with ATC (thissequence encodes an improved hydroxynitrile lyase wherein the alanineresidue at position 2 is substituted with isoleucine);

The sequence as shown in SEQ ID NO: 103 wherein the nucleotides GCA atpositions 4 to 6 are substituted with AGA, AGQ CGA, CGC, CGG or CGT,preferably the nucleotides GCA at positions 4 to 6 are substituted withCGT (this sequence encodes an improved hydroxynitrile lyase wherein thealanine residue at position 2 is substituted with arginine);

The sequence as shown in SEQ ID NO: 103 wherein the nucleotides GCA atpositions 4 to 6 are substituted with CAA or CAG, preferably thenucleotides GCA at positions 4 to 6 are substituted with CAG (thissequence encodes an improved hydroxynitrile lyase wherein the alanineresidue at position 2 is substituted with glutamine);

The sequence as shown in SEQ ID NO: 103 wherein the nucleotides GCA atpositions 4 to 6 are substituted with CCA, CCC, CCG or CCT, preferablythe nucleotides G and A at positions 4 and 6 are substituted with C andG, respectively (this sequence encodes an improved hydroxynitrile lyasewherein the alanine residue at position 2 is substituted with proline);

The sequence as shown in SEQ ID NO: 103 wherein the nucleotides GCA atpositions 4 to 6 are substituted with ACA, ACC, ACG or ACT, preferablythe nucleotides G and A at positions 4 and 6 are substituted with A andC, respectively (this sequence encodes an improved hydroxynitrile lyasewherein the alanine residue at position 2 is substituted withthreonine);

The sequence as shown in SEQ ID NO: 103 wherein the nucleotides GCA atpositions 4 to 6 are substituted with TAC or TAT, preferably thenucleotides GCA at positions 4 to 6 are substituted with TAC (thissequence encodes an improved hydroxynitrile lyase wherein the alanineresidue at position 2 is substituted with tyrosine);

The sequence as shown in SEQ ID NO: 103 wherein the nucleotides GCA atpositions 4 to 6 are substituted with TTA, TTG, CTA, CTC, CTG or CTT,preferably the nucleotides GCA at positions 4 to 6 are substituted withCTG (this sequence encodes an improved hydroxynitrile lyase wherein thealanine residue at position 2 is substituted with leucine);

The sequence as shown in SEQ ID NO: 103 wherein the nucleotides GCA atpositions 4 to 6 are substituted with ATG (this sequence encodes animproved hydroxynitrile lyase wherein the alanine residue at position 2is substituted with methionine);

The sequence as shown in SEQ ID NO: 103 wherein the nucleotides GCA atpositions 4 to 6 are substituted with AGC, AGT, TCA, TCC, TCG or TCT,preferably the nucleotides GCA at positions 4 to 6 are substituted withAGC (this sequence encodes an improved hydroxynitrile lyase wherein thealanine residue at position 2 is substituted with serine);

The sequence as shown in SEQ ID NO: 103 wherein the nucleotides GCA atpositions 4 to 6 are substituted with GAA or GAG, preferably thenucleotide C at position 5 is substituted with A (this sequence encodesan improved hydroxynitrile lyase wherein the alanine residue at position2 is substituted with glutamic acid);

The sequence as shown in SEQ ID NO: 103 wherein the nucleotides GCA atpositions 4 to 6 are substituted with GGA, GGC, GGG or GGT, preferablythe nucleotides C and A at positions 5 and 6 are substituted with G andC, respectively (this sequence encodes an improved hydroxynitrile lyasewherein the alanine residue at position 2 is substituted with glycine);

The sequence as shown in SEQ ID NO: 103 wherein the nucleotides GCA atpositions 4 to 6 are substituted with GAC or GAT, preferably thenucleotides C and A at positions 5 and 6 are substituted with A and C,respectively (this sequence encodes an improved hydroxynitrile lyasewherein the alanine residue at position 2 is substituted with asparticacid);

(B)

The sequence as shown in SEQ ID NO: 2 or 103 wherein the nucleotides CACat positions 307 to 309 are substituted with ATG (this sequence encodesan improved hydroxynitrile lyase wherein the histidine residue atposition 103 is substituted with methionine);

The sequence as shown in SEQ ID NO: 2 or 103 wherein the nucleotides CACat positions 307 to 309 are substituted with TTA, TTG, CTA, CTC, CTG orCTT, preferably the nucleotides A and C at positions 308 and 309 aresubstituted with T and G, respectively (this sequence encodes animproved hydroxynitrile lyase wherein the histidine residue at position103 is substituted with leucine);

The sequence as shown in SEQ ID NO: 2 or 103 wherein the nucleotides CACat positions 307 to 309 are substituted with ATA, ATC or ATT, preferablythe nucleotides C and A at positions 307 and 308 are substituted with Aand T, respectively (this sequence encodes an improved hydroxynitrilelyase wherein the histidine residue at position 103 is substituted withisoleucine);

The sequence as shown in SEQ ID NO: 2 or 103 wherein the nucleotides CACat positions 307 to 309 are substituted with GTA, GTC, GTG or GTT,preferably the nucleotides C and A at positions 307 and 308 aresubstituted with G and T, respectively (this sequence encodes animproved hydroxynitrile lyase wherein the histidine residue at position103 is substituted with valine);

The sequence as shown in SEQ ID NO: 2 or 103 wherein the nucleotides CACat positions 307 to 309 are substituted with TGC or TGT, preferably thenucleotides C and A at positions 307 and 308 are substituted with T andG, respectively (this sequence encodes an improved hydroxynitrile lyasewherein the histidine residue at position 103 is substituted withcysteine);

The sequence as shown in SEQ ID NO: 2 or 103 wherein the nucleotides CACat positions 307 to 309 are substituted with CAA or CAG, preferably thenucleotide C at position 309 is substituted with G (this sequenceencodes an improved hydroxynitrile lyase wherein the histidine residueat position 103 is substituted with glutamine);

The sequence as shown in SEQ ID NO: 2 or 103 wherein the nucleotides CACat positions 307 to 309 are substituted with AGC, AGT, TCA, TCC, TCG orTCT, preferably the nucleotides CAC at positions 307 to 309 aresubstituted with TCG (this sequence encodes an improved hydroxynitrilelyase wherein the histidine residue at position 103 is substituted withserine);

The sequence as shown in SEQ ID NO: 2 or 103 wherein the nucleotides CACat positions 307 to 309 are substituted with ACA, ACC, ACG or ACT,preferably the nucleotides CAC at positions 307 to 309 are substitutedwith ACG (this sequence encodes an improved hydroxynitrile lyase whereinthe histidine residue at position 103 is substituted with threonine);

The sequence as shown in SEQ ID NO: 2 or 103 wherein the nucleotides CACat positions 307 to 309 are substituted with GCA, GCC, GCG or GCT,preferably the nucleotides C and A at positions 307 and 308 aresubstituted with G and C, respectively (this sequence encodes animproved hydroxynitrile lyase wherein the histidine residue at position103 is substituted with alanine);

The sequence as shown in SEQ ID NO: 2 or 103 wherein the nucleotides CACat positions 307 to 309 are substituted with TGG (this sequence encodesan improved hydroxynitrile lyase wherein the histidine residue atposition 103 is substituted with tryptophan);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides CAT atpositions 307 to 309 are substituted with ATG (this sequence encodes animproved hydroxynitrile lyase wherein the histidine residue at position103 is substituted with methionine);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides CAT atpositions 307 to 309 are substituted with TTA, TTG, CTA, CTC, CTG orCTT, preferably the nucleotides A and T at positions 308 and 309 aresubstituted with T and C, respectively (this sequence encodes animproved hydroxynitrile lyase wherein the histidine residue at position103 is substituted with leucine);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides CAT atpositions 307 to 309 are substituted with ATA, ATC or ATT, preferablythe nucleotides CAT at positions 307 to 309 are substituted with ATC(this sequence encodes an improved hydroxynitrile lyase wherein thehistidine residue at position 103 is substituted with isoleucine);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides CAT atpositions 307 to 309 are substituted with GTA, GTC, GTG or GTT,preferably the nucleotides CAT at positions 307 to 309 are substitutedwith GTC (this sequence encodes an improved hydroxynitrile lyase whereinthe histidine residue at position 103 is substituted with valine);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides CAT atpositions 307 to 309 are substituted with TGC or TGT, preferably thenucleotides C and A at positions 307 and 308 are substituted with T andQ respectively (this sequence encodes an improved hydroxynitrile lyasewherein the histidine residue at position 103 is substituted withcysteine);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides CAT atpositions 307 to 309 are substituted with CAA or CAG, preferably thenucleotide T at positions 309 is substituted with G (this sequenceencodes an improved hydroxynitrile lyase wherein the histidine residueat position 103 is substituted with glutamine);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides CAT atpositions 307 to 309 are substituted with AGC, AGT, TCA, TCC, TCG orTCT, preferably the nucleotides CAT at positions 307 to 309 aresubstituted with AGC (this sequence encodes an improved hydroxynitrilelyase wherein the histidine residue at position 103 is substituted withserine);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides CAT atpositions 307 to 309 are substituted with ACA, ACC, ACG or ACT,preferably the nucleotides CAT at positions 307 to 309 are substitutedwith ACC (this sequence encodes an improved hydroxynitrile lyase whereinthe histidine residue at position 103 is substituted with threonine);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides CAT atpositions 307 to 309 are substituted with GCA, GCC, GCG or GCT,preferably the nucleotides CAT at positions 307 to 309 are substitutedwith GCC (this sequence encodes an improved hydroxynitrile lyase whereinthe histidine residue at position 103 is substituted with alanine);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides CAT atpositions 307 to 309 are substituted with TGG (this sequence encodes animproved hydroxynitrile lyase wherein the histidine residue at position103 is substituted with tryptophan).

It should be noted here that the improved hydroxynitrile lyase gene ofthe present invention also includes genes encoding improvedhydroxynitrile lyases in which a histidine residue at a neighboringposition to position 103 is substituted with another amino acid asdescribed in (II-2) above (i.e., improved hydroxynitrile lyases in whichthe histidine residue corresponding to the histidine residue at position103 of cassava- or Para rubber tree-derived hydroxynitrile lyase ismutated).

(C)

The sequence as shown in SEQ ID NO: 2 wherein the nucleotides AAG atpositions 526 to 528 are substituted with CCA, CCC, CCG or CCT,preferably the nucleotides AAG at positions 526 to 528 are substitutedwith CCC (this sequence encodes an improved hydroxynitrile lyase whereinthe lysine residue at position 176 is substituted with proline);

The sequence as shown in SEQ ID NO: 2 wherein the nucleotides AAG atpositions 595 to 597 are substituted with CCA, CCC, CCG or CCT,preferably the nucleotides AAG at positions 595 to 597 are substitutedwith CCC (this sequence encodes an improved hydroxynitrile lyase whereinthe lysine residue at position 199 is substituted with proline);

The sequence as shown in SEQ ID NO: 2 wherein the nucleotides AAA atpositions 670 to 672 are substituted with CCA, CCC, CCG or CCT,preferably the nucleotides AAA at positions 670 to 672 are substitutedwith CCC (this sequence encodes an improved hydroxynitrile lyase whereinthe lysine residue at position 224 is substituted with proline);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides AAA atpositions 526 to 528 are substituted with CCA, CCC, CCG or CCT,preferably the nucleotides AAA at positions 526 to 528 are substitutedwith CCC (this sequence encodes an improved hydroxynitrile lyase whereinthe lysine residue at position 176 is substituted with proline);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides AAA atpositions 595 to 597 are substituted with CCA, CCC, CCG or CCT,preferably the nucleotides AAA at positions 595 to 597 are substitutedwith CCC (this sequence encodes an improved hydroxynitrile lyase whereinthe lysine residue at position 199 is substituted with proline);

The sequence as shown in SEQ ID NO: 3 wherein the nucleotides AAA atpositions 670 to 672 are substituted with CCA, CCC, CCG or CCT,preferably the nucleotides AAA at positions 670 to 672 are substitutedwith CCT (this sequence encodes an improved hydroxynitrile lyase whereinthe lysine residue at position 224 is substituted with proline);

The sequence as shown in SEQ ID NO: 103 wherein the nucleotides AAG atpositions 523 to 525 are substituted with CCA, CCC, CCG or CCT,preferably the nucleotides AAG at positions 523 to 525 are substitutedwith CCC (this sequence encodes an improved hydroxynitrile lyase whereinthe lysine residue at position 175 is substituted with proline);

The sequence as shown in SEQ ID NO: 103 wherein the nucleotides AAG atpositions 592 to 594 are substituted with CCA, CCC, CCG or CCT,preferably the nucleotides AAG at positions 592 to 594 are substitutedwith CCC (this sequence encodes an improved hydroxynitrile lyase whereinthe lysine residue at position 198 is substituted with proline);

The sequence as shown in SEQ ID NO: 103 wherein the nucleotides AAA atpositions 667 to 669 are substituted with CCA, CCC, CCG or CCT,preferably the nucleotides AAA at positions 667 to 669 are substitutedwith CCT (this sequence encodes an improved hydroxynitrile lyase whereinthe lysine residue at position 223 is substituted with proline).

The improved hydroxynitrile lyase genes of (D) to (G) are improvedhydroxynitrile lyase genes which have any two or all three of theembodiments described in (A) to (C) above.

For example, (D) is a combination of (A) and (B). Thus, as one exampleof the gene of (D), a gene may be given which is an improvedhydroxynitrile lyase gene having the sequence as shown in SEQ ID NO: 2wherein the nucleotides G and T at positions 4 and 5 are substitutedwith A and A, respectively, and the nucleotide CAC at positions 307 to309 are substituted with ATG.

Further, the improved hydroxynitrile lyase gene of the present inventionmay also have any of the following substitutions in addition to theembodiments described in (A) to (G) above.

The sequence as shown in SEQ ID NO: 2 wherein the nucleotides TTT atpositions 373 to 375 are substituted with CTT (this sequence encodes animproved hydroxynitrile lyase wherein the phenylalanine residue atposition 125 is substituted with leucine);

The sequence as shown in SEQ ID NO: 2 wherein the nucleotides ACC atpositions 436 to 438 are substituted with ACA;

The sequence as shown in SEQ ID NO: 2 wherein the nucleotides ACC atpositions 613 to 615 are substituted with TCC (this sequence encodes animproved hydroxynitrile lyase wherein the phenylalanine residue atposition 205 is substituted with leucine); or

The sequence as shown in SEQ ID NO: 2 wherein the nucleotides GAT atpositions 703 to 705 are substituted with GGT (this sequence encodes animproved hydroxynitrile lyase wherein the aspartic acid residue atposition 235 is substituted with glycine).

Further, the improved hydroxynitrile lyase gene of the present inventionalso includes a DNA which hybridizes to a DNA consisting of a nucleotidesequence complementary to the nucleotide sequence described in any oneof (A) to (G) above under stringent conditions, and encodes a proteinwhose hydroxynitrile lyase activity per transformant is higher than thatof the corresponding wild-type hydroxynitrile lyase. Such a DNA may beobtained from a cDNA library or genomic library by a known hybridizationmethod, such as colony hybridization, plaque hybridization or Southernblotting, using as a probe an improved hydroxynitrile lyase gene DNAconsisting of the nucleotide sequence described in any one of (A) to (G)above or a complementary sequence thereto, or a fragment thereof. Thelibrary may be one prepared by a known method. Alternatively, acommercial cDNA library or genomic library may also be used.

The term “stringent conditions” refers to conditions at the time ofwashing after hybridization. Specifically, it means conditions wheresalt concentration is 300-2000 mM and temperature is 40-75° C.,preferably salt concentration is 600-900 mM and temperature is 65° C.Specific example of stringent conditions includes 2×SSC and 50° C. Oneof ordinary skill in the art can appropriately select the saltconcentration and temperature of the buffer, and other conditions suchas the concentration and length of the probe, reaction time, etc. tothereby decide conditions for obtaining a DNA encoding the improvedhydroxynitrile lyase of the present invention.

Detailed procedures of hybridization methods are described, for example,in Molecular Cloning, A Laboratory Manual 2nd ed. (Cold Spring HarborLaboratory Press (1989)). As an example of the above DNA whichhybridizes, a DNA comprising a nucleotide sequence having 40% or more,preferably 60% or more, more preferably 90% or more identity with thenucleotide sequence as described in any one of (A) to (G) above or apartial fragment thereof may be given.

In the present invention, the method for preparing the improvedhydroxynitrile lyase gene may be any of known methods for introducingmutations. Usually, the improved hydroxynitrile lyase gene may beprepared by a known method. For example, a method in which asite-directed substitution is introduced into a wild-type hydroxynitrilelyase gene using a commercial kit; a method in which a genetic DNA isselectively cleaved and then selected oligonucleotides are removed/addedand ligated; or the like may be enumerated.

These site-directed mutagenesis methods are described, for example, inMolecular Cloning, A Laboratory Manual 2nd ed. (Cold Spring Harbor Press(1989)); Current Protocols in Molecular Biology (John Wiley & Sons(1987-1997)); Kunkel, Proc. Natl. Acad. Sci. USA 82: 488-92 (1985);Kramer and Fritz, Method. Enzymol. 154:350-67 (1987); Kunkel, Method.Enzymol. 85: 2763-6 (1988). Recently, mutagenesis introduction kitsutilizing site-directed mutagenesis based on the Kunkel method or thegapped duplex method may be used to perform mutagenesis. Examples ofsuch kits include QuickChange™ Site-Directed Mutagenesis Kit(Stratagene), GeneTailor™ Site-Directed Mutagenesis System (Invitrogen)and TaKaRa Site-Directed Mutagenesis System (e.g., Mutan-K, Mutan-SuperExpress Km; Takara Bio). When a site into which a mutation of interestis to be introduced is located near a restriction enzyme site (wheredigestion and ligation are easy) in the target gene sequence, a geneticDNA fragment introduced the mutation of interest may be obtained easilyby performing PCR using primers (synthetic oligo-DNAs) into which themutation of interest is introduced. Alternatively, improved hydroxylnitrile lyase genes may be obtained as a synthetic gene by PCRelongation with a combination of synthetic oligo-DNAs (assembly PCR).

It is also possible to obtain improved hydroxynitrile lyase genes fromwild-type hydroxynitrile lyase genes by random mutation introductionmethods, e.g., contacting/reacting mutagens such as hydroxylamine ornitrous acid; mutagenize by UV irradiation; introducing random mutationsby PCR (polymerase chain reaction), etc.

(IV) Recombinant Vector and Transformant

(IV-1) Recombinant Vector

In order to express the thus obtained improved hydroxynitrile lyase geneof the present invention in a host, an expression cassette into which atranscription promoter upstream of the gene and a terminator downstreamof the gene are inserted may be constructed and inserted into anexpression vector. Alternatively, when an expression vector into whichthe improved hydroxynitrile lyase gene is to be introduced already has atranscription promoter and a terminator, the mutated gene may beinserted between the promoter ant the terminator without construction ofan expression cassette. For introduction of the improved hydroxynitrilelyase gene into a vector, such methods as using restriction enzymes or atopoisomerase may be used. If necessary for the introduction, anappropriate linker may be added. In the present invention, it is alsopossible to perform such integrating operations concurrently withoperations for preparing the improved hydroxynitrile lyase gene.Briefly, using primers which have a nucleotide sequence substituted withanother nucleotide sequence to encode other amino acids and, as atemplate, a recombinant vector into which the wild-type hydroxynitrilelyase gene has been cloned, PCR may be performed and the resultantamplified product may be integrated into a vector.

The type of promoter is not particularly limited as long as it allowsappropriate expression of the gene of interest in a host. Specificexamples of promoters useful in the present invention include, but arenot limited to, E. coli-derived tryptophan operon (trp) promoter andlactose operon (lac) promoter; lambda phage-derived PL promoter and PRpromoter; Bacillus subtilis-derived gluconate synthase promoter (gnt),alkali protease promoter (apr), neutral protease promoter (npr) andα-amylase promoter (amy). Modified and designed sequences such as tacpromoter and trc promoter may also be used.

Terminators are not necessarily needed, and the type thereof is notparticularly limited. For example, ρ factor non-dependent terminatorssuch as lipoprotein terminator, trp operon terminator or rrnB terminatormay be used.

As important nucleotide sequences for translation into amino acids,ribosome binding sequences such as SD sequence and Kozak sequence areknown. These sequences may be inserted upstream of the mutated gene.When a prokaryote is used as a host, SD sequence may be added by PCR orthe like. When a eukaryote is used as a host, Kozak sequence may beadded. Examples of SD sequence include E. coli-derived or B.subtilis-derived sequences. However, any SD sequence may be used as longas it functions in a desired host such as E. coli or B. subtilis. Forexample, a consensus sequence consisting of 4 or more consecutivenucleotides complementary to a 3′ terminal region of 16S ribosome RNAmay be synthesized by DNA synthesis and used.

Generally, vectors comprise an element to select a transformant ofinterest (selective marker). Specific examples of selective markersinclude, but are not limited to, drug resistance genes, auxotrophiccomplementary genes and genes that render assimilability, and they areappropriately selected considering the purpose or the host to be used.For example, specific examples of drug resistance genes used asselective marker in E. coli include ampicillin resistance gene,kanamycin resistance gene, dihydrofolate reductase gene, and neomycinresistance gene.

The vector to be used in the present invention is not particularlylimited as long as it retains the above-described mutated gene. A vectorsuitable for each host may be used. Specific examples of vectors usefulin the present invention include plasmid DNA, bacteriophages DNA,retrotransposon DNA and artificial chromosomal DNA. For example, when E.coli is used as a host, a vector comprising a region capable ofautonomous replication in E. coli, such as pTrc99A (Centraalbureau voorSchimmelcultures (CBS), Netherland; http://www.cbs.knaw.nl/), pUC19(Takara Bio; Japan), pKK233-2 (Centraalbureau voor Schimmelcultures(CBS), Netherland; http://www.cbs.knaw.nl/), pET-12 (Novagen; Germany)or pET-26b (Novagen; Germany) may be used. If necessary, a vectormodified from these vectors may be used. It is also possible to use anexpression vector with high expression efficiency (such as pTrc99A orpKK233-2 having trc promoter and lac operator) may be used.

A recombinant vector comprising the above-described improvedhydroxynitrile lyase gene is included within the scope of the presentinvention.

(IV-1) Transformant

Transformants or transductants (sometimes, these are collectivelyreferred to as the “transformant”) may be prepared by transforming ortransducing the recombinant vector of the present invention into a host.The transformant is also included in the scope of the present invention.

The host to be used in the present invention is not particularly limitedas long as it is capable of expressing an improved hydroxynitrile lyaseof interest upon introduction of the above-described recombinant vector.Specific examples of hosts to be used in the present invention include,but are not limited to, bacteria such as Escherichia coli and Bacillussubtilis, yeasts (Pichia, Saccharomyces), fungi (Aspergillus), animalcells, insect cells and plant cells.

When a bacterium is used as a host, especially preferable in the presentinvention is E. coli. Examples of E. coli strains to be used in thepresent invention include, but are not limited to, K12 strain and Bstrain, and derivatives from these wild-type strains, such as JM109strain, XL1-Blue strain and C600 strain. Especially when a lactoseoperon (lac) promoter mentioned above or a derivative promoter therefromis used as an expression promoter, expression of a gene of interestbecomes inducible by IPTG or the like in a host with lacI repressorgene; and expression of a gene of interest becomes permanent in a hostwithout lacI repressor gene. So, a convenient host may be selected.These strains are easily available, for example, from American TypeCulture Collection (ATCC). As a bacillus, Bacillus subtilis may be used,for example. The method for introducing a recombinant vector into thebacterium is not particularly limited. Any method of DNA transfer intobacteria may be used, e.g., the method using calcium ion orelectroporation.

When a yeast is used as a host, Saccharomyces cerevisiae,Schizosaccharomyces pombe, Pichia pastoris or the like may be used. Themethod for introducing a recombinant vector into the yeast is notparticularly limited. Any method of DNA transfer into yeasts may beused, e.g., electroporation, the spheroplast method, and the lithiumacetate method.

When an animal cell is used as a host, simian COS-7 cells, Vero cells,CHO cells, mouse L cells, rat GH3 cells, human FL cells or the like maybe used. As the method for introducing a recombinant vector into theanimal cell, electroporation, the calcium phosphate method orlipofection may be used, for example.

When an insect cell is used as a host, Sf9 cells, Sf21 cells or the likemay be used. As the method for introducing a recombinant vector into theinsect cell, the calcium phosphate method, lipofection orelectroporation may be used, for example.

When a plant cell is used as a host, cells to be used in the presentinvention include, but are not limited to, tobacco BY-2 cells. As themethod for introducing a recombinant vector to the plant cell, theagrobacterium method, the particle gun method, the PEG method orelectroporation may be used, for example.

(V) Method for Preparing Culture and Improved Hydroxynitrile Lyase

In the present invention, an improved hydroxynitrile lyase may beprepared by culturing the above-described transformant and recoveringthe improved hydroxynitrile lyase from the resultant culture.

The present invention also includes a method for producing an improvedhydroxynitrile lyase, characterized by recovering the improvedhydroxynitrile lyase from the culture.

In the present invention, the term “culture” means any of the followingmaterials: culture supernatant, cultured cells, cultured microorganisms,or disrupted materials from cells or microorganisms. The cultureobtained by culturing the transformant of the present invention isincluded in the scope of the present invention.

Cultivation of the transformant of the present invention is carried outin accordance with conventional methods commonly used for culturinghosts. The improved hydroxynitrile lyase of interest is accumulated inthe above-described culture.

As a medium to culture the transformant of the present invention, eithera natural or synthetic medium may be used as long as it contains carbonsources, nitrogen sources, inorganic salts, etc. assimilable by the hostand is capable of efficient culture of the transformant. As carbonsources, carbohydrates such as glucose, galactose, fructose, sucrose,raffinose and starches; organic acids such as acetic acid and propionicacid; and alcohols such as ethanol and propanol may be enumerated. Asnitrogen sources, ammonia; ammonium salts of inorganic or organic acidssuch as ammonium chloride, ammonium sulfate, ammonium acetate andammonium phosphate; and other nitrogen-containing compounds may beenumerated. Further, peptone, yeast extract, meat extract, corn steepliquor, and various amino acids may also be used. As inorganicsubstances, potassium dihydrogenphosphate, dipotassiumhydrogenphosphate, magnesium phosphate, magnesium sulfate, sodiumchloride, iron(III) sulfate, manganese sulfate, zinc sulfate, coppersulfate, calcium carbonate and the like may be enumerated. If necessary,defoaming agents may be added to prevent foaming during cultivation.Vitamins or the like may also be added optionally. If necessary,antibiotics such as ampicillin or tetracycline may be added duringcultivation.

The cultivation may be performed under selective pressure in order toprevent the falling off of the vector and the gene of interest. Briefly,when the selective marker is a drug resistance gene, the relevant drugmay be added to the medium; and when the selective marker is anauxotrophic complementary gene, the relevant nutritional factor may beremoved from the medium. Further, when the selective marker is a genethat renders assimilability, the relevant assimilation factor may beadded as the sole factor, if necessary. For example, when E. colitransformed with a vector comprising an ampicillin resistance gene iscultured, ampicillin may be added to the medium during cultivation, ifnecessary.

When a transformant transformed with an expression vector having aninducible promoter is cultured, an inducer may be added to the medium,if necessary. For example, when a transformant transformed with anexpression vector having a promoter inducible byisopropyl-β-D-thiogalactoside (IPTG), IPTG or the like may be added tothe medium. When a transformant transformed with an expression vectorhaving a trp promoter inducible by indoleacetic acid (IAA), IAA or thelike may be added to the medium.

Cultivation conditions for transformants are not particularly limited.Any conditions may be used as long as they do not inhibit theproductivity of the improved hydroxynitrile lyase of interest and thegrowth of the host. Usually, cultivation temperature is 10-45° C.,preferably 10-40° C., more preferably 15-40° C., and still morepreferably 20-37° C. If necessary, the temperature may be changed duringcultivation. Time of cultivation is 5-120 hours, preferably 5-100 hours,more preferably 10-100 hours, and still more preferably 15-80 hours.Adjustment of pH is performed with an inorganic or organic acid or analkali solution. When the transformant is E. coli, pH is adjusted to6-9. Examples of cultivation methods include solid culture, stationaryculture, shaking culture and aeration-agitation culture.

Especially when E. coli transformants are cultured, it is preferable toculture the transformants under aerobic conditions by shaking culture oraeration-agitation culture (in a jar fermenter). Although E. colitransformants may be cultured by conventional solid culture, it isstrongly recommended to employ liquid culture for them. As a medium toculture E. coli transformants, a medium containing one or more nitrogensources such as yeast extract, tryptone, polypeptone, corn steep liquor,a decoction of soybean or wheat bran; supplemented with one or moreinorganic salts such as sodium chloride, potassium dihydrogenphosphate,dipotassium hydrogenphosphate, magnesium sulfate, magnesium, chloride,iron(III) chloride, iron(III) sulfate or manganese sulfate; andoptionally supplemented with carbohydrate materials, vitamins or thelike; may be used. It is appropriate to adjust the initial pH of themedium to 7-9. The culture is performed at 5-40° C., preferably at10-37° C., for 5-100 hours. Preferably, aeration-agitation submergedculture, shaking culture, stationary culture, feeding culture or thelike is used. Especially when an improved hydroxynitrile lyase isproduced in an industrial scale, aeration-agitation culture may be used.Operational method for aeration-agitation culture is not particularlylimited. Any of batch culture, fed-batch culture (semi-batch culture) orcontinuous culture may be used. In particular, when enhancement ofproductivity per apparatus, time, cost or operation is intended by highdensity culture, fed-batch culture may be used. The composition of fedmedium used in fed-batch culture may be the same as that of the initial(batch) medium. Alternatively, the composition may be altered but it ispreferable that the medium component concentrations are higher thanthose in the initial medium. The volume of fed medium is notparticularly limited. Usually, ½ or less volume relative to the initialmedium may be added. Examples of the feeding mode for fed mediuminclude, but are not limited to, constant feeding, exponential feeding,stepwise increase feeding, specific growth-rate control feeding, pH-statfeeding, DO-stat feeding, glucose concentration control feeding, acetateconcentration monitoring feeding, and fuzzy neural network feeding(Trends in Biotechnology (1996), 14, 98-105). Any of these feeding modesmay be used as long as a desired hydroxynitrile lyase productivity isachieved. When fed-batch culture is practiced, the time of culturetermination is not necessarily limited to the time when addition of fedmedium has been completed. If necessary, culture may be continuedfurther and stopped at the time when hydroxynitrile lyase activity pertransformant is the highest.

As a medium to culture a transformant obtained from an animal cell as ahost, commonly used RPMI1640 medium, DMEM medium or one of these mediumsupplemented with fetal bovine serum, etc. may be used. Usually,cultivation is performed under 5% CO₂ at 37° C. for 1 to 30 days. Duringcultivation, antibiotics such as kanamycin or penicillin may be added tothe medium, if necessary. When the transformant (or transductant) is aplant cell or plant tissue, cultivation may be performed using aconventional plant cell culture medium such as MS basal medium or LSbasal medium. The culture method may be either conventional solidculture or liquid culture.

When the transformant is a plant cell or plant tissue, cultivation maybe performed using a conventional plant cell culture medium such as MSbasal medium or LS basal medium. The culture method may be eitherconventional solid culture or liquid culture.

By culturing the transformant under the conditions as described above,it is possible to allow accumulation of the improved hydroxynitrilelyase of the present invention in the resultant culture, that is, any ofthe following materials: culture supernatant, cultured cells, culturedmicroorganisms, or disrupted cells or microorganisms.

When the improved hydroxynitrile lyase is produced in microorganisms orcells, the improved hydroxynitrile lyase of interest may be recovered bydisrupting microorganisms or cells after cultivation.

Before disruption, if necessary, solid-liquid separation operations suchas centrifugation or membrane filtration may be performed for mediumremoval and washing.

Centrifuge is not particularly limited as long as it can provide acentrifugal force to precipitate microorganisms or cells. A cylindertype or disc-stack type centrifuge may be used. As the centrifugalforce, 500 G-20,000 G may be used.

The membrane which may be used in this process may be either amicrofiltration (MF) membrane or ultrafiltration (UF) membrane as longas it can achieve the intended solid-liquid separation. Usually, it ispreferable to use a microfiltration (MF) membrane. Microfiltration maybe classified into dead-end mode or cross-flow (tangential flow) modebased on the direction of flow; into weight mode, pressure mode, vacuummode and centrifugal mode based on the addition of pressure; and intorotary mode and continuous mode based on the operational method. Any ofthese modes may be used as long as solid-liquid separation can beachieved. The materials of MF membrane may be roughly divided intopolymer, ceramic, metal and a combination thereof. The material is notparticularly limited provided that it does not decrease the activity ofimproved hydroxynitrile lyase and the recovery ratio of that activity atthe time of solid-liquid separation. Preferably, a polymer membrane madeof polysulfone, polyethersulfone, polytetrafluoroethylene,polyvinylidine fluoride, polyvinyl chloride, polypropylene, polyolefin,polyethylene, polycarbonate, polyacrylonitrile, mixed cellulose ester,cuprammonium regenerated cellulose ester, polyimide, nylon, teflon orthe like may be used. With respect to the pore size of membrane, anysize which is capable of capturing microorganisms or cells and capableof concentration operation may be used. Usually, the pore size may bearound 0.1-0.5 μm.

In the present invention, the term “activity recovery ratio” means therelative ratio (%) of the activity recovered after an operation such assolid-liquid separation, taking the activity before the operation as100%.

At the time of solid-liquid separation by centrifugation and membranefiltration, water, buffer or isotonic solution may be added to performdilution washing, if necessary. The buffer used for this purpose is notparticularly limited provided that it does not decrease the activity ofimproved hydroxynitrile lyase and the activity recovery ratio thereof atsolid-liquid separation. For example, a buffer whose salt concentrationis 5-500 mM, preferably about 5-150 mM and pH is about 4-8 may be used.As components of the buffer, salts including phosphoric acid salts,citric acid salts and acetic acid salts of sodium or potassium may beenumerated. Specific examples of buffers include 5 mM potassiumphosphate buffer (pH 6-7) and 20 mM sodium acetate buffer (pH 5-6). Asthe isotonic solution, 0.7-0.9% sodium chloride solution may be used,for example. Further, substances capable of stabilizing the improvedhydroxynitrile lyase (e.g., flavonoids) may be added (Food Technologyand Biotechnology (2001), 39(3), 161-167).

As the method for disrupting microorganisms or cells, sonication, highpressure treatment with a French press or homogenizer, grindingtreatment with a bead mill, collision treatment with an impact crusher,enzymatic treatment using such as lysozyme, cellulase or pectinase,freeze-thaw treatment, treatment with a hypotonic solution, cell lysisinduction by phage, etc. may be enumerated. Any of these methods may beused alone or in combination. When microorganisms or cells are disruptedin an industrial scale, use of high pressure treatment, grindingtreatment or collision treatment is preferable from the viewpoints ofeasiness in operation, recovery ratio and cost. Occasionally, thesephysical crushing operations may be combined with enzymatic treatment.Operational conditions for individual disrupting methods are notparticularly limited provided that the recovery ratio of improvedhydroxynitrile lyase from microorganisms or cells is sufficiently high.The “sufficiently high recovery ratio of improved hydroxynitrile lyase”means preferably 85% or more, more preferably 90% or more, still morepreferably 95%, and most preferably 99% or more.

When grinding treatment is performed with a bead mill, for example,beads 2.5-6.0 g/cm³ in density and 0.1-1.0 mm in size may be packed inthe mill at about 80-85%. Either batch method or continuous method maybe used for the operation. The concentration of microorganisms or cellsis not particularly limited. For example, the concentration may be about6-12% for a bacterium and about 14-18% for a yeast.

When high pressure treatment is performed, the pressure to be added isnot particularly limited provided that the recovery ratio of improvedhydroxynitrile lyase from microorganisms or cells is sufficiently high.For example, crushing may be performed with a pressure of about 40-150MPa. The concentration of microorganisms or cells is not particularlylimited. For example, the concentration may be about 20% or less. Ifnecessary, multi-stage treatment may be performed by connectingapparatuses in series or using an apparatus with multi-stage structure,to thereby improve the efficiency of crushing and operation. Usually, atemperature increase of 2-3° C. per 10 MPa of pressure occurs. So, it ispreferable to perform cooling treatment when necessary.

When collision treatment is performed, microorganisms or a cell slurryto be crushed are subjected to spray rapid freezing or the like(freezing rate: e.g., several thousand ° C./min) to obtain frozenmicroparticles (e.g., 50 μm or less), which are then stroked against acollision board by means of a high speed (e.g., 300 m/sec) carrier gas.Thus, microorganisms or cells are crushed.

As a result of the above-described disrupting treatment formicroorganisms or cells, intracellular nucleic acid flows out, whichincreases the viscosity of the treated liquid and may results indifficulty in handling it. In such a case, or when improvement ofactivity recovery ratio can be expected in a later step of residueseparation, nucleic acid removal or degradation may be performed todecrease the viscosity of the treated liquid or to improve the activityrecovery ratio in the step of residue separation, if necessary. As themethod for removing or degrading the nucleic acid in the liquidcontaining disrupted cells, any method may be used provided as long asit is capable of removing or degrading nucleic acid without decreasingthe activity of improved hydroxynitrile lyase or the recovery ratio ofthe activity. For example, as described on pages 200-201 in BiochemistryExperiment Course Vol. 5, a method in which protamine sulfate orstreptomycin is added to the liquid containing disrupted cells toprecipitate nucleic acid; a method in which nucleic acid is degradedwith a nuclease; a method in which liquid-liquid separation is performedwith dextran-polyethylene glycol; or the like may be enumerated. It mayalso be effective to add a physical crushing treatment further. Of thesemethods, the method of degradation using a nuclease may be employed whenrapid degradation of nucleic acid is desired while avoiding complicationof steps. The nuclease to be used in the nucleic acid degradationtreatment may be any nuclease as long as it acts on at leastdeoxyribonucleic acid (DNA), has the ability to catalyze nucleic aciddegradation and reduces the degree of DNA polymerization. It may bepossible to utilize a nuclease inherent within the transformant cell.Alternatively, it is possible to add an exogenous nuclease. Examples ofthe exogenous nuclease to be added include bovine spleen-derived DNase I(Takara Bio, Japan), porcine spleen-derived DNase II (Wako Purechemical,Japan), Serratia marcescens-derived Benzonase® Nuclease (Takara Bio,Japan) and Staphylococcus aureus-derived nuclease (Wako Purechemical,Japan). The amount of enzyme to be added varies depending on the type ofnuclease and the definition of unit (U), but one of ordinary skill inthe art can select appropriately. If necessary, cofactors such asmagnesium required for the nuclease may be added. The treatmenttemperature varies depending on the type of nuclease. When the nucleaseis derived from a mesophilic organism, temperatures ranging from 20 to40° C. may be use, for example.

When it is necessary to remove microorganism or cell debris from theliquid containing disrupted cells, such methods as centrifugation orfiltration (dead end mode or cross flow mode) may be used.

Centrifugal operations may be performed as described above. Whenmicroorganism or cell debris is too fine to precipitate easily, it ispossible to add a flocculant to improve the efficiency of debrisprecipitation. Organic polymer flocculants may be classified intocationic, anionic, amphoteric and nonionic flocculants based onionicity; and classified into acryl type, polyethyleneimine type,condensed polycation (polyamine) type, dimethyldiallylammonium chloridetype and chitosan type based on raw material. The flocculant to be usedin the present invention may be any flocculant provided that it does notdecrease the activity of improved hydroxynitrile lyase or the recoveryratio of the activity and yet is capable of improving the efficiency ofdebris separation. Examples of acrylic aqueous monomers which can becomponents of acryl type flocculants include acrylamide, sodiumacrylate, sodium acrylamide-2-methyl-propanesulfonate,dimethylaminoethyl-methacrylate,methacryloyloxyethyl-trimethylammonium-chloride,methacryloyloxyethyl-benzyldimethyl-ammonium chloride,dimethylaminoethyl-acrylate,acryloyloxyethyl-trimethylammonium-chloride,dimethylaminopropyl-acrylamide, acrylamidepropyl-trimethylammonium-chloride, and polyamidine-chloride. Singlepolymers of these monomers; copolymers of diversified composition ofthese monomers; and high molecular modified products of these monomers;are enumerated as acryl type flocculants. As representative examples ofcationic polymer flocculants, polyaminoalkylmethacrylates, copolymers ofpolyaminoalkylmethacrylate and acrylamide, Mannich modified products ofpolyacrylamide, polydimethyldiallyammonium salts, polyvinylimidazolines,polyacrylamides, amine type polycondensated products and the like may beenumerated, and a great number of them have already been commercialized.Major products include Sanpoly-K-601, K-602 (principal component:polyamine; Sankyo Kasei); Kuriflock LC-599 (principal components:polyamine and polyamide; Kurita Water); Hymolock M-166, M-566, M-966(principal component: acrylamide modified product; Kyoritu OrganicIndustry); Uniflocker UF-301, UF-304, UF-305 (principal component:polyacrylamide; Unitika), UF-330, UF-340 (principal component:aminomethacrylic acid ester; Unitika), UF-505 (principal component:dicyano amine; Unitika); Ryufloc C-110 (principal component: polyamine;Dainippon Ink & Chemicals); and Purifloc C-31 (principal component:polyamine; Dow Chemical) may be enumerated. Further, K-400 series,KM-200 series, KM-1200 series, KAM-200 series, KD-200 series, KP-000series, KP-100 series, KP-200 series, KP-300 series, KP-500 series,KP-1200 series, KA-000 series, KA-200 series, KA-300 series, KA-400series, KA-600 series, KA-700 series and KA-800 series manufactured byDiaNitrix (Japan) may also be enumerated. These flocculants may be usedalone or in a combination of two or more. Any of the above-describedflocculants may be used in the present invention provided that it doesnot decrease the activity of improved hydroxynitrile lyase and theactivity recovery ratio thereof and yet is capable of improving theefficiency of debris separation. Specifically, DiaNitrix (Japan)products such as K-401, K-403B, K-405, K-408, K-409, K-415, KP201H,KP309 or KP7000 may be used, for example. The amount of flocculant to beadded varies depending on the type of flocculant and the condition ofthe liquid containing disrupted microorganisms or cells. For example,the flocculant may be used at a concentration of 1/50-½, preferably1/20-⅕, relative to the dry weight % concentration of the disruptedmicroorganism. The flocculant may be added as follows, for example;after dissolved in water, the flocculant is added to the liquidcontaining disrupted microorganisms or cells, and then the liquid isleft stationary or agitated for at least 5 minutes to 24 hours,preferably for 30 minutes to 10 hours. The temperature at that time maybe preferably 0-60° C., more preferably 0-50° C., still more preferably0-40° C. When pH adjustment is necessary, an inorganic salt may be addedat a final concentration of 5-200 mM to buffer the liquid.Alternatively, a substance which stabilizes the improved hydroxynitrilelyase may be added.

When the debris is separated by filtration, either microfiltration (MF)membrane or ultrafiltration (UF) membrane may be used as along as thedesired separation of debris can be achieved. Usually, it is preferableto use a MF membrane. Any MF membrane may be used as long as it iscapable of separation of debris. The pore size of the membrane is notparticularly limited provided that the pore allows capturing of themicroorganism or cell debris and yet the activity of improvedhydroxynitrile lyase is recovered in the filtrate. For example, amembrane with a pore size of about 0.1 to 0.5 μm may be used. Further,with the use of a filter aid and occasionally a flocculant, a membraneor filter paper with a pore size of 0.5 μm or more may also be used.Specific examples of filter aids include diatomaceous earth, cellulosepowder and active carbon. Flocculants are as described above.

The supernatant obtained after removal of the debris is a cell extractsoluble fraction, and this may be used as a crude enzyme solutioncontaining the improved hydroxynitrile lyase. Subsequently, commonbiochemical methods used for isolation/purification of proteins, such asammonium sulfate precipitation, various chromatographies [e.g., gelfiltration chromatography (with Sephadex column, etc.), ion exchangechromatography (with DEAE-Toyopearl, etc.), affinity chromatography,hydrophobic chromatography (with butyl Toyopearl, etc.), anionchromatography (with MonoQ column, etc.)] or SDS polyacrylamide gelelectrophoresis may be performed independently or in a combination tothereby isolate/purify the hydroxynitrile lyase from the culture.

When the improved hydroxynitrile lyase is produced within themicroorganisms or cells, the microorganisms or cells per se may berecovered by centrifugation, membrane separation, etc. and used inenzyme reactions of interest without disrupting. In this case, it isalso possible to use the treated culture which include cultured cells ina gel such as acrylamide, cultured cells treated with glutaraldehyde, orcultured cells carried by an inorganic substance such as alumina,silica, zeolite, diatomaceous earth or the like, if necessary.

On the other hand, when the transformant of the present invention is agenetic recombinant and there is a possible risk of leakage of thetransformant into environments or mixing thereof with the final productduring the production process, or a possible risk of causing amicroorganism pollution secondarily due to inappropriate handling or thelike of the used transformant, inactivation of the transformant may beperformed. As the method of inactivation, any method may be usedprovided as long as it is capable of inactivating the transformantwithout decreasing the activity of improved hydroxynitrile lyase or therecovery ratio of the activity. For example, such methods as thermaltreatment, cell disrupting treatment or drug treatment may be performedindependently or in a combination. For example, the transformant may beinactivated by treating it with a drug before or after cell disruptingtreatment. The drug to be used varies depending on the type of host ofthe transformant. For example, cationic surfactants such as benzethoniumchloride, cetylpyridinium chloride, methylstearoyl chloride andcetyltrimethylammonium bromide; or aldehydes such as glutaraldehyde maybe enumerated. Further, alcohols such as ethanol, thiols such as2-mercaptoethanol, amines such as ethylenediamine, and amino acids suchas cysteine, ornithine and citrulline may also be enumerated. Theconcentration of drug may be a concentration which is capable ofinactivating the transformant without decreasing the activity ofimproved hydroxynitrile lyase or the recovery ratio of the activity. Forexample, when the host of the transformant is E. coli and the drugs arebenzethonium chloride and glutaraldehyde, respective finalconcentrations of these drugs are preferably in the range from0.05-0.5%. At the time of this inactivation treatment, flavonoids or thelike may be added in order to improve the stability of the improvedhydroxynitrile lyase. The treatment temperature may be 0-50° C.,preferably 0-40° C. The pH is preferably 4-8.

On the other hand, when the improved hydroxynitrile lyase is producedoutside the microorganisms or cells, the culture broth may be used as itis or after removal of microorganisms or cells by the above-describedcentrifugation or filtration. Then, the improved hydroxynitrile lyasemay be recovered from the culture by such method as extraction byammonium sulfate precipitation and, if necessary, purified by one or acombination of the following methods: dialysis and variouschromatographies such as gel filtration, ion exchange chromatography,affinity chromatography and the like.

When the transformant is a plant cell or tissue, cells are disrupted bylysis treatment using enzymes such as cellulase or pectinase, sonicationtreatment, or grinding treatment. Subsequently, if necessary, commonbiochemical methods used for isolation/purification of proteins, such asammonium sulfate precipitation, various chromatographies [e.g., gelfiltration chromatography (with Sephadex column, etc.), ion exchangechromatography (with DEAE-Toyopearl, etc.), affinity chromatography,hydrophobic chromatography (with butyl Toyopearl, etc.), anionchromatography (with MonoQ column, etc.)] or SDS polyacrylamide gelelectrophoresis may be performed independently or in a combination tothereby isolate/purify the hydroxynitrile lyase from the culture.

The thus obtained improved hydroxynitrile lyase is included in the scopeof the present invention. The production yield of the improvedhydroxynitrile lyase may be calculated by measuring the activity ofimproved hydroxynitrile lyase per unit, such as culture equipment,culture broth, cell wet weight or dry weight, weight of protein inenzyme solution, etc. The unit is not particularly limited. The activityof improved hydroxynitrile lyase may be expressed by the value of eitherdegradation activity or synthesis activity as described earlier.Alternatively, the activity may be calculated indirectly by means ofanalysis such as SDS-PAGE. One of ordinary skill in the art can performSDS-PAGE using known methods. Further, it is also possible to calculatethe activity by preparing an antibody to the improved hydroxynitrilelyase and using immunological techniques such as Western blotting orELISA.

In the present invention, it is also possible to collect the improvedhydroxynitrile lyase from the above-described improved hydroxynitrilelyase gene or a recombinant vector comprising the gene. Briefly, it ispossible to produce the improved hydroxynitrile lyase using a cell-freeprotein synthesis system without using living cells. The term “cell-freeprotein synthesis system” means a system which synthesizes a proteinusing a cell extract in an artificial container such as a test tube. Acell-free transcription system which synthesizes RNA from DNA as atemplate is also included in the cell-free protein synthesis system usedin the present invention. In this case, an organism corresponding to theabove-described host is the organism from which the cell extractdescribed below is derived. As the cell extract, a eukaryote- orprokaryote-derived cell extract (e.g., an extract from wheat germs or E.coli) may be used. These cell extracts may be either concentrated ornon-concentrated. Cell extracts may be obtained by such methods asultrafiltration, dialysis or polyethylene glycol (PEG) precipitation. Inthe present invention, cell-free protein synthesis may also be performedwith a commercial kit. Examples of such kits include reagent kitsPROTEIOS™ (Toyobo) and TNT™ System (Promega) and synthesis apparatusesPG-Mate™ (Toyobo) and RTS (Roche Diagnostics).

The improved hydroxynitrile lyase obtained by the above-describedcell-free synthesis may be purified, for example, by appropriatelyselecting chromatography.

(VI) Method for Preparing Cyanohydrin and Method for PreparingHydroxycarboxylic Acid

The improved hydroxynitrile lyase prepared as described above may beutilized in the production of substances as an enzyme catalyst. Forexample, by contacting the above-described improved hydroxynitrile lyasewith either a ketone compound or aldehyde compound and a cyanidecompound, an optically active cyanohydrin may be prepared. As the enzymecatalyst, a culture obtained by culturing a host into which the improvedhydroxynitrile lyase gene has been transferred as to express the gene inany hosts or a treated culture may be used. The treated culture, forexample, include cultured cells in a gel such as acrylamide, culturedcells treated with glutaraldehyde, or cultured cells carried by aninorganic carrier such as alumina, silica, zeolite or diatomaceous earthmay be enumerated.

Either a ketone compound or aldehyde compound and a cyanide compoundwhich are used as substrates are selected considering the substratespecificity of the enzyme and the stability of the enzyme againstsubstrates. When the enzyme is a cassava (Manihot esculenta)-derivedhydroxynitrile lyase, suitable substrates are an aldehyde compound and acyanide compound. As the aldehyde compound, benzaldehyde is preferable.As the cyanide compound, prussic acid is preferable.

The effect of metal ions in cyanohydrin synthesis reaction becomes lesswhen H103M improved hydroxynitrile lyase is used, as compared to thecase where wild-type hydroxynitrile lyase is used. Further, thesynthesis of cyanohydrin is hardly affected by the presence of nickelions.

The form of use and the reaction mode of a biocatalyst are selectedappropriately depending on the type, etc. of the biocatalyst. As theform of use of the biocatalyst, the above-described culture or purifiedenzyme may be used as it is. Alternatively, the culture or purifiedenzyme may be carried on an appropriate carrier and used as animmobilized enzyme.

The method of reaction and the method of recovering cyanohydrin aftercompletion of the reaction are selected appropriately considering theproperties of the substrates and the enzyme catalyst. It is preferableto recycle an enzyme catalyst as long as it is not deactivated. From theviewpoints of preventing deactivation and facilitating recycling, it ispreferable to use the enzyme catalyst in the form of a treated culture.

In the present invention, the term “optically active” refers to thestate of a substance where one enantiomer is contained more than theother enantiomer, or the state of a substance where the substance isconsisting of either one of the enantiomers.

It is also possible to convert the recovered optically activecyanohydrin to optically active hydroxycarboxylic acid by performinghydrolysis reaction with a mineral acid such as sulfuric acid orhydrochloric acid. Preferably, the hydrolysis reaction is performedusing a mineral acid in the same manner as in conventional methods.Examples of mineral acid which may be used here include hydrochloricacid, sulfuric acid, nitric acid, boric acid, phosphoric acid andperchloric acid. Of these, hydrochloric acid is preferable. Usually, thesolvent used in the hydrolysis process is water. If necessary, polarsolvents such as dimethylsulfoxide, dimethylformamide ordimethylacetamide; hydrocarbon solvents such as toluene, hexane orheptane; or ether solvents such as diethyl ether, diisopropyl ether,t-butylmethyl ether or tetrahydrofuran may be used in conjunction withwater. These solvents may be used alone or in a combination. The amountof mineral acid to be used is preferably 0.5-20 equivalents, morepreferably 0.9-10 equivalents and most preferably 1-5 equivalents,relative to the amount of optically active cyanohydrin contained in thereaction mixture supplied to hydrolysis. Use of a mineral acid withinthis range is preferable because it is advantageous economically andimproves the recovery ratio of hydroxycarboxylic acid. The reactiontemperature of the hydrolysis process is preferably from −5° C. to theboiling point of the solvent used, especially preferably in the rangefrom 10 to 90° C. The temperature within this range is desirable fromthe viewpoints of the rate of hydrolysis reaction and reduction ofimpurities.

Hereinbelow, the present invention will be described more specificallywith reference to the following Examples. However, the present inventionis not limited by these Examples.

Example 1 Obtainment of Plant Codon Wild-Type Hydroxynitrile Lyase Gene

(1) Preparation of Plant Codon Wild-Type Hydroxynitrile Lyase Gene byPCR

Based on the nucleotide sequence disclosed in GenBank accession numberZ29091, a cassava (Manihot esculenta)-derived hydroxynitrile lyase generepresented by the sequence of SEQ ID NO: 2 was synthesized by PCR.

Specifically, 20 oligonucleotides designated F01-F10 and R01-R10 (SEQ IDNOS: 4-23) were designed and synthesized. Briefly, the 20oligonucleotides F01-F10 and R01-R10 were designed so that they arecomplementary to a nucleotide sequence comprising the cassava (Manihotesculenta)-derived hydroxynitrile lyase gene disclosed in GenBankaccession number Z29091 and its 5′ and 3′ untranslated regions (sensestrand) and a complementary sequence thereto (antisense strand) (1,041bp). In F01, 11 nucleotides (shown in italics) containing a BamHIrecognition site are added to its 5′ end. In R01, 9 nucleotides (shownin italics) containing a KpnI recognition site are added to its 5′ end.These oligonucleotides were designed so that the 1,041 bp comprising thecassava-derived hydroxynitrile lyase gene are ultimately amplified.These oligonucleotides were designed so that they overlap with theadjacent oligonucleotide(s) by about 20 bp. For example, F01 and F02 aredesigned so that the underlined part of F01 and the underlined part ofF02 are overlapping with each other. FIG. 1 schematically illustratesthe positional relations among these 20 oligonucleotides F01-F10 andR01-R10 (SEQ ID NOS: 4-23).

F01 (SEQ ID NO: 4): cgggatccccaaaaagagttagatatcatttccaaaatggtaactgcacattttgttctgattcataccatt F02 (SEQ ID NO: 5):ttgttctgattcataccatttgccatggtgcatggatttggcataagctc aaaccagcccttgagagagcF03 (SEQ ID NO: 6): aaaccagcccttgagagagctggccacaaagtcactgcactggacatggcagccagcggcattgacccaa F04 (SEQ ID NO: 7):agccagcggcattgacccaaggcaaattgagcagattaattcatttgatg aatactctgaacccttattgF05 (SEQ ID NO: 8): tattgccagacaccgttcatagcccatcttacactgtggaaaagcttttggagtcgtttcctgactggag F06 (SEQ ID NO: 9):gagtcgtttcctgactggagagacacagagtattttacgttcactaatat cactggagagacaattacaaF07 (SEQ ID NO: 10): cactggagagacaattacaacaatgaagctgggcttcgtacttctgagggaaaatttatttaccaaatgc F08 (SEQ ID NO: 11):atcaagacaaaatatttttaccagactttcaacgctggcaaattgcaaac tacaaaccagacaaggtttaF09 (SEQ ID NO: 12): tacaaaccagacaaggtttatcaggttcaaggtggagatcataagctccagcttacaaaaactgaggagg F10 (SEQ ID NO: 13):gcttacaaaaactgaggaggtagctcatattctccaagaggtggctgatg catatgcttgaagcttttagR01 (SEQ ID NO: 14): gcggtacccttaataggatatttatttatttaatttaaagattacataatagggataacattcccttaaatacacacat R02 (SEQ ID NO: 15):attcccttaaatacacacatctcagcaaatgaagagacaccaacgtggaactctcccatatttaaagaaaaaaaaactca R03 (SEQ ID NO: 16):tttaaagaaaaaaaaactcaaactttattttagtgcaatttaattctcac atgaaaatgtgagattatttR04 (SEQ ID NO: 17): atgaaaatgtgagattatttataactgcacccaggttaacttaataggagctaaaagcttcaagcatatg R05 (SEQ ID NO: 18):taaaaatattttgtcttgatcggtccaaatataaactttcttaattgatc cgtaacctttttcggtgaacR06 (SEQ ID NO: 19): cgtaacctttttcggtgaacttcggtctctgagccaaaacattttgaaacagtgatcccttcctcattac R07 (SEQ ID NO: 20):agtgatcccttcctcattaccatttttgccagttcatattccccatcagt gcatttggtaaataaattttR08 (SEQ ID NO: 21): atgaacggtgtctggcaataaggaattgtggaaaacaccagctgcaattttgtcaacgtatctatcagca R09 (SEQ ID NO: 22):tgtcaacgtatctatcagcagcaatagcaatattcagccctgcacagctc tcaccaacaatgatgaccttR10 (SEQ ID NO: 23): tcaccaacaatgatgaccttttccccttgagggagtttctccaagaaagtcaataagggttcagagtatt

Each of the oligonucleotides was lyophilized and re-suspended indistilled water to give a concentration of 100 pmol/μl. One μl aliquotwas taken from each of the 20 oligonucleotide suspensions to prepare amix oligo. The resultant mixture was added to PCR-mix (Pwo 10× buffer,dNTP mix, Pwo DNA polymerase) (Boehringer Mannheim). Table 3 shows thecomposition of the PCR reaction solution.

TABLE 3 PCR solution A (μl) B (μl) C (μl) Mix oligo (μl) 0.5 1 2 Pwo 10× buffer 5 5 5 dNTP mix 5 5 5 Pwo DNA polymerase 0.5 0.5 0.5 Distilledwater 39 38.5 37.5

PCR was performed for 55 cycles (94° C. for 30 sec, 52° C. for 30 secand 72° C. for 30 sec) to extend the oligonucleotides to therebysynthesize the gene of interest (1st PCR).

Subsequently, the thus synthesized gene was amplified (2nd PCR).Briefly, to 1.3 μl of the reaction product of PCR solution B in the 1stPCR, 5 μl of Pwo 10× buffer, 5 μl of dNTP mix, 0.5 μl of Pwo DNApolymerase, 36.2 μl of distilled water and 1 μl of external primers wereadded. As the external primers, F01(SEQ ID NO: 4) and R01(SEQ ID NO: 14)were used. The 2nd PCR were performed for 23 cycles (94° C. for 30 sec,50° C. for 30 sec and 72° C. for 60 sec) to amplify the gene.

The amplified product of the 2nd PCR was confirmed on 1.5% agarose gel.

(2) Preparation of Recombinant Vector Comprising Plant Codon Wild-TypeHydroxynitrile Lyase Gene

A 0.9 kb band which is the amplified product in the 2nd PCR obtained in(1) above was purified with QIAquick Gel Extraction Kit (QIAGEN). TheDNA purified from gel (5 μl) was digested with restriction enzymes BamHI(1 μl) (its recognition site is included in oligonucleotide F01) andKpnI (1 μl) (its recognition site is included in oligonucleotide R01) at37° C. for one hour. Then, the DNA was purified from the reactionsolution by phenol extraction, chloroform extraction and ethanolprecipitation [Molecular Cloning, A Laboratory Manual, 2nd ed. (ColdSpring Harbor Laboratory Press (1989))]. The thus purified DNA (5 μl), avector pUC19 (Takara Bio) (1 μl) predigested with BamHI and KpnI,distilled water (4 μl) and solution I (DNA Ligation Kit ver.2; TakaraBio) (10 μl) were mixed to prepare a ligation mixture. This mixture wasincubated at 16° C. for 12 hours to thereby ligate the amplified productto the vector.

(3) Preparation of E. coli JM109 Competent Cells

E. coli JM109 strain was inoculated into 1 ml of LB medium (1% Bactotryptone, 0.5% Bacto yeast extract, 0.5% NaCl) and precultured at 37° C.for 5 hours aerobically. The resultant preculture (0.4 ml) was added to40 ml of SOB medium (2% Bacto tryptone, 0.5% Bacto yeast extract, 10 mMNaCl, 2.5 mM KCl, 1 mM MgSO₄, 1 mM MgCl₂) and cultured at 18° C. for 20hours. The resultant culture was harvested by centrifugation (3,700×g,10 minutes, 4° C.) and, after addition of 13 ml of cold TF solution (20mM PIPES-KOH (pH 6.0), 200 mM KCl, 10 mM CaCl₂, 40 mM MnCl₂) thereto,the culture was left at 0° C. for 10 minutes. Subsequently, the culturewas re-centrifuged (3,700×g, 10 minutes, 4° C.) to remove thesupernatant. The precipitated E. coli cells were suspended in 3.2 ml ofcold TF solution and, after addition of 0.22 ml of dimethylsulfoxidethereto, the cell suspension was left at 0° C. for 10 minutes.

(4) Cloning of the Plant Codon Wild-Type Hydroxynitrile Lyase Gene

The competent cell prepared in (3) above (200 μl) was added to theligation product prepared in (2) above (10 μl) and left at 0° C. for 30minutes. Subsequently, heat shock was given to the competent cell at 42°C. for 30 sec, followed by cooling at 0° C. for 2 minutes. Then, 1 ml ofSOC medium (20 mM glucose, 2% Bacto tryptone, 0.5% Bacto yeast extract,10 mM NaCl, 2.5 mM KCl, 1 mM MgSO₄, 1 mM MgCl₂) was added thereto, andthe resultant mixture was subjected to shaking culture at 37° C. for 1hour. The resultant culture was plated in 200 μl aliquots on LB Amp agarmedium (LB medium containing 100 mg/L ampicillin and 1.5% agar) andcultured overnight at 37° C. A plurality of transformant colonies grownon the agar medium were cultured overnight in 1.5 ml of LB Amp medium(LB medium containing 100 mg/L ampicillin) at 37° C. Each of theresultant cultures was harvested, followed by recovery of therecombinant vector with Flexi Prep (Amersham Bioscience). The nucleotidesequence of the resultant recombinant vector was analyzed with CEQ DTCSQuick Start Kit and a fluorescence sequencer CEQ 2000XL DNA Analysissystem (both from BECKMAN COULTER; USA). As primers, oligonucleotidesF01-F10 and R01-R10 were used. One of the recombinant vectors having anucleotide sequence identical to the nucleotide sequence of the cassava(Manihot esculenta)-derived hydroxynitrile lyase gene as disclosed inGenBank accession number Z29091 and shown in SEQ ID NO: 2 was designatedpUME.

Example 2 Preparation of Plant Codon Wild-Type Hydroxynitrile LyaseExpression Vectors

(1) Preparation of Plant Codon Wild-Type Hydroxynitrile Lyase ExpressionVector (Based on pUC19)

An SD sequence was added to the nucleotide sequence of the plant codonwild-type hydroxynitrile lyase gene obtained in Example 1 to obtain aDNA fragment, which was then inserted at the PstI-BamHI site of pUC19 tothereby prepare a pUC19-based plant codon wild-type hydroxynitrile lyaseexpression vector pUMESD, as described below. First, a modified DNAfragment encoding the hydroxynitrile lyase was prepared by PCR. Thereaction mixture for PCR was composed of 5 μl of Pwo 10× buffer, 5 μl ofdNTP mix, 0.5 μl of Pwo DNA polymerase, 36.2 μl of distilled water, 1 μlof sense and antisense primers, and 1 μl of pUME as a template. PCR wasperformed as follows: 95° C. for 2 minutes for denaturation, 30 cyclesof 94° C. for 30 sec, 50° C. for 30 sec and 72° C. for 2 minutes; andfinally 72° C. for 10 minutes. The sense primer MES-1 (SEQ ID NO: 24)consists of 62 nucleotides, and has an PstI recognition site, a ribosomebinding site, the TAG termination codon for the lacZ gene frame of pUC19and the ATG initiation codon for the hydroxynitrile lyase gene in itssequence. The antisense primer MES-2 (SEQ ID NO: 25) consists of 33nucleotides and has a BamHI recognition site in its sequence.

Sense primer: (SEQ ID NO: 24)ccccaaactgcagtaaggaggaatagaaaatggtaactgcacattttgtt ctgattcataccAntisense primer: (SEQ ID NO: 25) tagtgcaattggatcctcacatgaaaatgtgag

The amplified PCR product obtained by the PCR was digested with PstI andBamHI, separated by agarose gel electrophoresis, and then purified withQIAquick Gel Extraction Kit. The resultant purified DNA fragment (5 μl),pUC19 (Takara Bio, Japan) (5 μl) predigested with PstI and BamHI, andsolution I (DNA Ligation Kit ver.2; Takara Bio) (10 μl) were mixed toprepare a ligation mixture. This mixture was incubated at 16° C. for 12hours to ligate the linker to the vector. E. coli JM109 strain wastransformed in the same manner as described in (4) in Example 1. Therecombinant vectors were recovered from the grown colonies. A plasmid inwhich the modified DNA fragment (PstI-BamHI fragment) comprising theplant codon wild-type hydroxynitrile lyase gene was inserted correctlydownstream of the lac promoter of pUC19 was designated expression vectorpUMESD.

(2) Preparation of Plant Codon Wild-Type Hydroxynitrile Lyase ExpressionVector (Based on pKK233-2)

A DNA fragment of the cassava (Manihot esculenta)-derived wild-typehydroxynitrile lyase gene obtained in Example 1 was inserted at theNcoI-Sse8387I site of pKK233-2(+Sse), a derivative of pKK233-2(Centraalbureau voor Schimmelcultures (CBS), Netherlands;http://www.cbs.knaw.nl/), to prepare a plant codon wild-typehydroxynitrile lyase expression vector pOXN103 based on pKK233-2, asdescribed below. First, a DNA fragment encoding the plant codonwild-type hydroxynitrile lyase was amplified by PCR in such a mannerthat the amplified fragment has at its both ends a restriction enzymerecognition site which can be easily introduced into an expressionvector. The PCR reaction mixture was composed of 5 μl of Pwo 10× buffer,5 μl of dNTP mix, 0.5 μl of Pwo DNA polymerase, 36.2 μl of distilledwater, 1 μl of sense and antisense primers, and 1 μl of plasmid pUME asa template. PCR was performed as follows: 95° C. for 2 minutes fordenaturation and 30 cycles of 94° C. for 30 sec, 50° C. for 30 sec and72° C. for 2 minutes. The sense primer OXYN-6 (SEQ ID NO: 26) consistsof 29 nucleotides, and includes an NcoI recognition site and the AGTinitiation codon and subsequent several codons of the hydroxynitrilelyase gene in its sequence. The antisense primer OXYN-9 (SEQ ID NO: 27)consists of 33 nucleotides, and includes an Sse8387I recognition site inits sequence.

(SEQ ID NO: 26) OXYN-6: ccaccatggtaactgcacattttgttctg (SEQ ID NO: 27)OXYN-9: ggcctgcaggttaacttaataggagctaaaagc

The resultant amplified PCR product was digested with Sse83871 and thenpartially digested with NcoI. The digest was separated by agarose gelelectrophoresis. A band (about 0.8 kb) comprising the full length of theplant codon wild-type hydroxynitrile lyase gene was cut out from thegel. The amplified PCR product in the gel was purified with QIAquick GelExtraction Kit.

Subsequently, an expression vector pKK233-2(+Sse) was prepared asdescribed below. pKK233-2 (Centraalbureau voor Schimmelcultures (CBS),Netherlands; http://www.cbs.knaw.nl/) (5 μl) was digested with HindIII(1 μl) and purified by phenol extraction, chloroform extraction andethanol precipitation. The purified digest was blunt-ended using DNABlunting Kit (Takara Bio). The thus treated solution was re-purified byphenol extraction, chloroform extraction and ethanol precipitation. Thepurified expression vector (5 μl) was subjected to dephosphorylationtreatment using Shrimp Alkaline Phosphatase (Takara Bio). The treatedsolution was re-purified by ethanol precipitation. The purified vectorDNA (5 μl), annealed Sse8387I phosphorylated linker pSse8387I (TakaraBio) (5 μl) and solution I (DNA Ligation Kit ver.2; Takara Bio) (10 μl)were mixed to prepare a ligation mixture. This mixture was incubated at16° C. for 12 hours to ligate the linker to the vector. E. coli JM109strain was transformed in the same manner as described in (4) inExample 1. The recombinant vectors were recovered from the growncolonies. The recovered recombinant vectors were subjected to Sse8387Idigestion, and those which had been confirmed to be digested linearlywere designated pKK233-2(+Sse). After digestion with restriction enzymesNcoI and Sse8387I, pKK233-2(+Sse) was purified by phenol extraction,chloroform extraction and ethanol precipitation.

The above-described DNA fragment (5 μl) of the plant codon wild-typehydroxynitrile lyase gene and expression vector pKK233-2(+Sse) (5 μl)were mixed. Solution I (DNA Ligation Kit ver.2; Takara Bio) (10 μl) wasadded to this mixture to prepare a ligation mixture. This mixture wasincubated at 16° C. for 12 hours to ligate the linker to the vector. E.coli JM109 strain was transformed in the same manner as described in (4)in Example 1. The recombinant vectors were recovered from the growncolonies. A plasmid in which the DNA fragment of the plant codonwild-type hydroxynitrile lyase gene was ligated to the vector correctlywas confirmed, and designated plant codon wild-type hydroxynitrile lyaseexpression recombinant vector pOXN103. At the same time, a plant codonwild-type hydroxynitrile lyase-expressing transformant JM109/pOXN103 wasobtained.

Example 3 Obtainment of E. coli Codon Wild-Type Hydroxynitrile LyaseGene

(1) Design and Preparation of E. coli Codon Wild-Type HydroxynitrileLyase Gene by PCR

A hydroxynitrile lyase gene was newly designed, and some of its codonswere converted to those codons frequently used in E. coli. Specifically,30 oligonucleotides Nos. 1-30 (of these, 6 types consisting of 49 nt; 21types consisting of 50 nt; 2 types consisting of 27 nt; and 1 typeconsisting of 48 nt) (SEQ ID NOS: 28-57) were designed and synthesizedultimately, and then prepared on 50 nmol scale. These 30oligonucleotides were designed so that they overlap by 20 nt (FIG. 2).

No. 1: (SEQ ID NO: 28) aaaagagttagatatcatttccaaaatggtgaccgcgcattttgtgctgNo. 2: (SEQ ID NO: 29)tccacgcgccatggcaaatggtatgaatcagcacaaaatgcgcggtcacc No. 3:(SEQ ID NO: 30) ttgccatggcgcgtggatttggcataaactgaaaccggcgctggaacgcgNo. 4: (SEQ ID NO: 31)ccatatccagcgcggtcactttatggcccgcgcgttccagcgccggtttc No. 5:(SEQ ID NO: 32) agtgaccgcgctggatatggcggcgagcggcattgatccgcgccagattgNo. 6: (SEQ ID NO: 33)cgctatattcatcaaagctgttaatctgttcaatctggcgcggatcaatg No. 7:(SEQ ID NO: 34) cagctttgatgaatatagcgaaccgctactgacctttctggaaaaactgcNo. 8: (SEQ ID NO: 35)cgcccacaataatcactttttcgccctgcggcagtttttccagaaaggtc No. 9:(SEQ ID NO: 36) aaaagtgattattgtgggcgaaagctgcgcgggcctgaacattgcgattgNo. 10: (SEQ ID NO: 37)ccgcaattttatccacatagcgatccgccgcaatcgcaatgttcaggccc No. 11:(SEQ ID NO: 38) ctatgtggataaaattgcggcgggcgtttttcataacagcctgctgccggNo. 12: (SEQ ID NO: 39)ccacggtatagctcgggctatgcacggtatccggcagcaggctgttatg No. 13:(SEQ ID NO: 40) tagcccgagctataccgtggaaaaactgctggaaagctttccggattggcNo. 14: (SEQ ID NO: 41)tgttggtaaaggtaaaatattcggtatcgcgccaatccggaaagctttcc No. 15:(SEQ ID NO: 42) atattttacctttaccaacattaccggcgaaaccattaccaccatgaaacNo. 16: (SEQ ID NO: 43) acaggttttcgcgcagcagcacaaagcccagtttcatggtggtaatggNo. 17: (SEQ ID NO: 44)ctgctgcgcgaaaacctgtttaccaaatgcaccgatggcgaatatgaac No. 18:(SEQ ID NO: 45) ggctgcctttgcgcatcaccattttcgccagttcatattcgccatcggtgNo. 19: (SEQ ID NO: 46)ggtgatgcgcaaaggcagcctgtttcagaacgtgctggcgcagcgcccg No. 20:(SEQ ID NO: 47) taatgctgccatagcctttttcggtaaatttcgggcgctgcgccagcacgNo. 21: (SEQ ID NO: 48) aaaaggctatggcagcattaaaaaagtgtatatttggaccgatcaggNo. 22: (SEQ ID NO: 49)agcgctgaaaatccggcagaaaaattttatcctgatcggtccaaatatac No. 23:(SEQ ID NO: 50) gccggattttcagcgctggcagattgcgaactataaaccggataaagtgNo. 24: (SEQ ID NO: 51)gtttatgatcgccgccctgcacctgatacactttatccggtttatagttc No. 25:(SEQ ID NO: 52) gggcggcgatcataaactgcagctgaccaaaaccgaagaagtggcgc No. 26:(SEQ ID NO: 53) catacgcatccgccacttcctgcagaatatgcgccacttcttcggttttgNo. 27: (SEQ ID NO: 54)agtggcggatgcgtatgcgtgaagcttttagctcctattaagttaacctg No. 28:(SEQ ID NO: 55) tgaaaatgtgagattatttataactgcacccaggttaacttaataggagcNo. 29: (SEQ ID NO: 56)taaataatctcacattttcatgtgagaattaaattgcactaaaataaag No. 30:(SEQ ID NO: 57) catatttaaagaaaaaaaaactcaaactttattttagtgcaatttaattc

Each of the oligonucleotides was lyophilized and re-suspended indistilled water to give a concentration of 100 pmol/μl. One μl aliquotwas taken from each of the 30 oligonucleotide suspensions to prepare amix oligo. The resultant mixture was added to PCR-mix (Pwo 10× buffer,dNTP mix, Pwo DNA polymerase) (Boehringer Mannheim) in the amountsindicated in Table 4 below.

TABLE 4 Composition of PCR Solution PCR solution A (μl) B (μl) Mix oligo0.5 1 Pwo 10 × buffer 5 5 dNTP mix 5 5 Pwo DNA polymerase 0.5 0.5Distilled water 39 38.5 Total (μl) 50 50

PCR was performed for 55 cycles (94° C. for 30 sec, 52° C. for 30 secand 72° C. for 30 sec) to extend the oligonucleotides to therebysynthesize a gene of interest (1st PCR).

Subsequently, the thus synthesized gene was amplified (2nd PCR).Briefly, to 1.3 μl of the reaction product of A or B shown in above theTable, 5 μl of Pwo 10× buffer, 5 μl of dNTP mix, 0.5 μl of Pwo DNApolymerase, 36.2 μl of distilled water and 1 μl of external primers wereadded. As the external primers, oligonucleotides No. 1 (SEQ ID NO: 28)and No. 30 (SEQ ID NO: 57) were used. The 2nd PCR were performed for 23cycles (94° C. for 30 sec, 50° C. for 30 sec and 72° C. for 60 sec) toamplify the gene.

The amplified product from the 2nd PCR was analyzed on 1.5% agarose gel.Subsequently, a 0.9 kb band was purified with QIAquick Gel ExtractionKit (QIAGEN). The DNA purified from gel (5 μl), vector pT7 Blue (1 μl),distilled water (4 μl) and solution I (DNA Ligation Kit ver.2; TakaraBio, Japan) (10 μl) were mixed to prepare a ligation mixture, which wasthen incubated at 16° C. for 12 hours to thereby ligate the amplifiedproduct to the vector. E. coli JM109 strain was transformed in the samemanner as described in (4) in Example 1. The recombinant vectors wererecovered from the grown colonies. A recombinant vector in which the DNAfragment of the E. coli codon wild-type hydroxynitrile lyase gene wasligated to the expression vector correctly was confirmed.

(2) Analysis of DNA Nucleotide Sequence

An automated plasmid separation apparatus (Kurabo, Osaka, Japan) wasused for preparing double-stranded DNA for use in sequencing. PlasmidDNA purified from the transformant obtained in (1) above were treatedwith EcoRI and XbaI, followed by analysis of the size of digested DNA onagarose gel. Plasmid No. 78 which showed a 0.9 kb DNA fragment was usedas a template DNA for determination of nucleotide sequences. Analysis ofnucleic acid sequences was performed with M13 Forward and Reverse IRD800Infrared Dye Labeled primer (ALOKA) by the dideoxynucleotide chaintermination method. Sequencing reaction was performed with ThermoSequence Cycle Sequencing Kit (Amersham Bioscience; Uppsala, Sweden) andthe reaction mixture was supplied to DNA Sequencer 4000L (Li-cor, Licon,Nebr., USA). It was confirmed that the genetic sequence of the E. colicodon wild-type hydroxynitrile lyase in plasmid No. 78 was identicalwith the genetic sequence of interest.

Example 4 Preparation of E. coli Codon Wild-Type Hydroxynitrile LyaseExpression Vector

An SD sequence was added to the nucleotide sequence of the E. coli codonwild-type hydroxynitrile lyase gene obtained in Example 3 to obtain aDNA fragment, which was then inserted at the SphI-BamHI site of pUC19 tothereby prepare a pUC19-based E. coli codon wild-type hydroxynitrilelyase expression vector pUMESDsy, as described below. First, a modifiedDNA fragment encoding the E. coli codon wild-type hydroxynitrile lyasewas prepared by PCR. The reaction mixture for PCR was composed of 5 μlof Pwo 10× buffer, 5 μl of dNTP mix, 0.5 μl of Pwo DNA polymerase, 36.2μl of distilled water, 1 μl of sense and antisense primers, and 1 μl ofplasmid No. 78 as a template. PCR was performed as follows: 95° C. for 2minutes for denaturation, and 30 cycles of 94° C. for 30 sec, 50° C. for30 sec and 72° C. for 2 minutes. The sense primer (SEQ ID NO: 58)consists of 61 nucleotides, and has an SphI recognition site, a ribosomebinding site, the TAG termination codon for the lacZ gene frame of pUC19and the ATG initiation codon for the hydroxynitrile lyase gene in itssequence. The antisense primer (SEQ ID NO: 59) consists of 37nucleotides and has a BamHI recognition site in its sequence.

Sense primer: (SEQ ID NO: 58)tgcaaagcatgctaaggaggaatagaaaatggtgaccgcgcattttgt gctgattcataccAntisense primer: (SEQ ID NO: 59) attttagtgcaattggatcctcacatgaaaatgtgag

The amplified PCR product obtained by PCR was digested with SphI andBamHI, separated by agarose gel electrophoresis, and then purified withQIAquick Gel Extraction Kit. This purified DNA fragment (5 μl), pUC19(Takara Bio, Japan) (5 μl) predigested with SphI and BamHI, and solutionI (DNA Ligation Kit ver.2; Takara Bio)(10 μl) were mixed to prepare aligation mixture. This mixture was incubated at 16° C. for 12 hours toligate the linker to the vector. E. coli JM109 strain was transformed inthe same manner as described in (4) in Example 1. The recombinantvectors were recovered from the grown colonies. A plasmid in which themodified DNA fragment (SphI-BamHI fragment) comprising the E. coli codonwild-type hydroxynitrile lyase gene was inserted correctly downstream ofthe lac promoter of pUC19 was designated expression vector pUMESDsy.

Example 5 Preparation of Expression Vector for Improved HydroxynitrileLyase in which the Amino Acid at Position 2 is Substituted, andHydroxynitrile Lyase-Expressing Transformant Comprising the Vector

(1) Preparation of Expression Vector for Improved Hydroxynitrile Lyasein which the Amino Acid at Position 2 is Substituted, and HydroxynitrileLyase-Expressing Transformant Comprising the Vector (Part 1)

Hydroxynitrile lyase genes in which the amino acid valine (Val; V) atposition 2 of the cassava (Manihot esculenta)-derived wild-typehydroxynitrile lyase is substituted with alanine (Ala; A), aspartic acid(Asp; D), glutamic acid (Glu; E), glycine (Gly; G), isoleucine (Ile; I),methionine (Met; M), threonine (Thr; T), asparagine (Asn; N), lysine(Lys; K), serine (Ser; S), phenylalanine (Phe; F), tyrosine (Tyr; Y),cysteine (Cys; C) or tryptophan (Trp; W) were prepared by introducing,at the time of PCR amplification, into a sense primer a restrictionenzyme recognition site (which is at the same time a codon encoding anamino acid of interest) capable of ligating to the NcoI restriction siteon the above-described expression vector pKK233-2(+Sse).

PCR reaction mixture was composed of 5 μl of Pwo 10× buffer, 5 μl ofdNTP mix, 0.5 μl of Pwo DNA polymerase, 36.2 μl of distilled water, 1 μlof sense and antisense primers, and 1 μl of pUME as a template. PCR wasperformed as follows: 95° C. for 2 minutes for denaturation, and 30cycles of 94° C. for 30 sec, 50° C. for 30 sec and 72° C. for 2 minutes.For sense primers, the following oligonucleotides were used.

OXYN-32: agaccatggc tactgcacat tttgtt (SEQ ID NO: 60; this sequenceconsists of 26 nucleotides, and has an NcoI recognition site and the ATGinitiation codon and subsequent several codons of the hydroxynitrilelyase gene; the codon corresponding to the amino acid at position 2 isGCT and encodes alanine.)OXYN-33: agaccatgga cactgcacat tttgtt (SEQ ID NO: 61; this sequenceconsists of 26 nucleotides, and has an NcoI recognition site and the ATGinitiation codon and subsequent several codons of the hydroxynitrilelyase gene; the codon corresponding to the amino acid at position 2 isGAC and encodes aspartic acid.)OXYN-34: agaccatgga aactgcacat tttgtt (SEQ ID NO: 62; this sequenceconsists of 26 nucleotides and has an NcoI recognition site and the ATGinitiation codon and subsequent several codons of the hydroxynitrilelyase gene; the codon corresponding to the amino acid at position 2 isGAA and encodes glutamic acid.)OXYN-35: agaccatggg cactgcacat tttgtt (SEQ ID NO: 63; this sequenceconsists of 26 nucleotides, and has an NcoI recognition site and the ATGinitiation codon and subsequent several codons of the hydroxynitrilelyase gene; the codon corresponding to the amino acid at position 2 isGGC and encodes glycine.)OXYN-10: atttccatca tgatcactgc acattttgtt ctg (SEQ ID NO: 64; thissequence consists of 33 nucleotides, and has a BspHI recognition siteand the ATG initiation codon and subsequent several codons of thehydroxynitrile lyase gene; the codon corresponding to the amino acid atposition 2 is ATC and encodes isoleucine.)OXYN-36: agatcatgat gactgcacat tttgttc (SEQ ID NO: 65; this sequenceconsists of 27 nucleotides, and has a BspHI recognition site and the ATGinitiation codon and subsequent several codons of the hydroxynitrilelyase gene; the codon corresponding to the amino acid at position 2 isATG and encodes methionine.)OXYN-37: agatcatgac cactgcacat tttgtt (SEQ ID NO: 66; this sequenceconsists of 26 nucleotides, and has a BspHI recognition site and the ATGinitiation codon and subsequent several codons of the hydroxynitrilelyase gene; the codon corresponding to the amino acid at position 2 isACC and encodes threonine.)OXYN-38: agatcatgaa cactgcacat tttgttc (SEQ ID NO: 67; this sequenceconsists of 27 nucleotides, and has a BspHI recognition site and the ATGinitiation codon and subsequent several codons of the hydroxynitrilelyase gene; the codon corresponding to the amino acid at position 2 isAAC and encodes asparagine.)OXYN-39: agatcatgaa aactgcacat tttgttc (SEQ ID NO: 68; this sequenceconsists of 27 nucleotides, and has a BspHI recognition site and the ATGinitiation codon and subsequent several codons of the hydroxynitrilelyase gene; the codon corresponding to the amino acid at position 2 isAAA and encodes lysine.)OXYN-40: agatcatgag cactgcacat tttgtt (SEQ ID NO: 69; this sequenceconsists of 26 nucleotides, and has a BspHI recognition site and the ATGinitiation codon and subsequent several codons of the hydroxynitrilelyase gene; the codon corresponding to the amino acid at position 2 isAGC and encodes serine.)OXYN-41: agaacatgtt cactgcacat tttgttc (SEQ ID NO: 70; this sequenceconsists of 27 nucleotides, and has a PciI recognition site and the ATGinitiation codon and subsequent several codons of the hydroxynitrilelyase gene; the codon corresponding to the amino acid at position 2 isTTC and encodes phenylalanine.)OXYN-42: agaacatgta cactgcacat tttgttc (SEQ ID NO: 71; this sequenceconsists of 27 nucleotides, and has a PciI recognition site and the ATGinitiation codon and subsequent several codons of the hydroxynitrilelyase gene; the codon corresponding to the amino acid at position 2 isTAC and encodes tyrosine.)OXYN-43: agaacatgtg cactgcacat tttgttc (SEQ ID NO: 72; this sequenceconsists of 27 nucleotides, and has a PciI recognition site and the ATGinitiation codon and subsequent several codons of the hydroxynitrilelyase gene; the codon corresponding to the amino acid at position 2 isTGC and encodes cysteine.)OXYN-44: agaacatgtg gactgcacat tttgttc (SEQ ID NO: 73; this sequenceconsists of 27 nucleotides, and has a PciI recognition site and the ATGinitiation codon and subsequent several codons of the hydroxynitrilelyase gene; the codon corresponding to the amino acid at position 2 isTGG and encodes tryptophan.)

As the antisense primer, OXYN-09 used in (2) in Example 2 was used.

The thus amplified PCR products were double-digested. For mutantssubstituted with alanine, aspartic acid, glutamic acid and glycine,restriction enzymes NcoI and Sse8387I were used. For mutants substitutedwith isoleucine, methionine, threonine, asparagine, lysine and serine,restriction enzymes BspHI and Sse8387I were used. For mutantssubstituted with phenylalanine, tyrosine, cysteine and tryptophan,restriction enzymes PciI and Sse8387I were used. It should be note thatdigestion with NcoI was partial digestion. After separation by agarosegel electrophoresis, a band comprising the full length hydroxynitrilelyase gene (about 0.8 kb) was purified with QIAquick Gel Extraction Kit.The purified DNA fragment of hydroxynitrile lyase gene (5 μl each) andthe expression vector pKK233-2(+Sse) (5 μl) prepared in (2) in Example 2were mixed. Solution I (DNA Ligation Kit ver.2; Takara Bio) (10 μl) wasfurther added to the mixture to prepare a ligation mixture. This mixturewas incubated at 16° C. for 12 hours to thereby ligate the linker to thevector. E. coli JM109 strain was transformed in the same manner asdescribed in (4) in Example 1. The recombinant vectors were recoveredfrom the grown colonies. Expression-type recombinant vectors in whichthe DNA fragment of the hydroxynitrile lyase gene was ligated to theexpression vector correctly were confirmed. The recombinant vectorscomprising genes encoding hydroxynitrile lyases in which the amino acidat position 2 is substituted with alanine, aspartic acid, glutamic acid,glycine, isoleucine, methionine, threonine, asparagine, lysine, serine,phenylalanine, tyrosine, cysteine and tryptophan were designatedpOXN103V2A, pOXN103V2D, pOXN103V2E, pOXN103V2G, pOXN103V21, pOXN103V2M,pOXN103V2T, pOXN103V2N, pOXN103V2K, pOXN103V2S, pOXN103V2F, pOXN103V2Y,pOXN103V2C and pOXN103V2W, respectively. At the same time, transformantsexpressing the individual hydroxynitrile lyases, respectively, were alsoobtained: JM109/pOXN103V2A, JM109/pOXN103V2D, JM109/pOXN103V2E,JM109/pOXN103V2G, JM109/pOXN103V21, JM109/pOXN103V2M, JM109/pOXN103V2T,JM109/pOXN103V2N, JM109/pOXN103V2K, JM109/pOXN103V2S, JM109/pOXN103V2F,JM109/pOXN103V2Y, JM109/pOXN103V2C and JM109/pOXN103V2W.

(2) Preparation of Expression Vector for Improved Hydroxynitrile Lyasein which the Amino Acid at Position 2 is Substituted, and HydroxynitrileLyase-Expressing Transformant Comprising the Vector (Part 2)

Hydroxynitrile lyase genes in which the amino acid valine (Val; V) atposition 2 of the cassava (Manihot esculenta)-derived wild-typehydroxynitrile lyase is substituted with arginine (Arg; R), glutamine(Gln; Q), histidine (His; H), leucine (Leu; L) or proline (Pro; P) wereprepared by site-directed mutagenesis with QuikChange™ Site-DirectedMutagenesis Kit (Stratagene) using the wild-type hydroxynitrile lyaseexpression vector pOXN103 prepared in (2) in Example 2 as a template.Primers used for introducing mutations were as follows.

Arginine Substitution Mutant

OXYN-45: aaacagacca tgcgtactgc acattttg (SEQ ID NO: 74; this is a senseprimer consisting of 28 nucleotides and has a complementary sequence toOXYN-46; the codon corresponding to the amino acid at position 2 is CGTand encodes arginine.)

OXYN-46: caaaatgtgc agtacgcatg gtctgttt (SEQ ID NO: 75; this is anantisense primer consisting of 28 nucleotides and has a complementarysequence to OXYN-45.)

Glutamine Substitution Mutant

OXYN-47: aaacagacca tgcagactgc acattttg (SEQ ID NO: 76; this is a senseprimer consisting of 28 nucleotides and has a complementary sequence toOXYN-48; the codon corresponding to the amino acid at position 2 is CAGand encodes glutamine.)

OXYN-48: caaaatgtgc agtctgcatg gtctgttt (SEQ ID NO: 77; this is anantisense primer consisting of 28 nucleotides and has a complementarysequence to OXYN-47.)

Histidine Substitution Mutant

OXYN-49: aaacagacca tgcacactgc acattttg (SEQ ID NO: 78; this is a senseprimer consisting of 28 nucleotides and has a complementary sequence toOXYN-50; the codon corresponding to the amino acid at position 2 is CACand encodes histidine.)

OXYN-50: caaaatgtgc agtgtgcatg gtctgttt (SEQ ID NO: 79; this is anantisense primer consisting of 28 nucleotides and has a complementarysequence to OXYN-49.)

Leucine Substitution Mutant

OXYN-51: aaacagacca tgctgactgc acattttg (SEQ ID NO: 80; this is a senseprimer consisting of 28 nucleotides and has a complementary sequence toOXYN-52; the codon corresponding to the amino acid at position 2 is CTGand encodes leucine.)

OXYN-52: caaaatgtgc agtcagcatg gtctgttt (SEQ ID NO: 81; this is anantisense primer consisting of 28 nucleotides and has a complementarysequence to OXYN-51.)

Proline Substitution Mutant

OXYN-53: aaacagacca tgccgactgc acattttg (SEQ ID NO: 82; this is a senseprimer consisting of 28 nucleotides and has a complementary sequence toOXYN-54; the codon corresponding to the amino acid at position 2 is CCGand encodes proline.)

OXYN-54: caaaatgtgc agtcggcatg gtctgttt (SEQ ID NO: 83; this is anantisense primer consisting of 28 nucleotides and has a complementarysequence to OXYN-53.)

According to the protocol of the above-mentioned Kit, extension reactionand DpnI treatment were performed. Then, E. coli JM109 was transformed,followed by recovery of plasmid DNA from grown colonies. Plasmids inwhich the DNA fragment of the hydroxynitrile lyase gene was ligated tothe expression vector correctly were confirmed. Expression vectorscomprising the genes encoding hydroxynitrile lyases in which the aminoacid at position 2 is substituted with arginine, glutamine, histidine,leucine and proline were designated pOXN103V2R, pOXN103V2Q, pOXN103V2H,pOXN103V2L and pOXN103V2P, respectively. At the same time, transformantsJM109/pOXN103V2R, JM109/pOXN103V2Q, JM109/pOXN103V2H, JM109/pOXN103V2Land JM109/pOXN103V2P expressing the individual hydroxynitrile lyaseswere also obtained.

Example 6 Evaluation of Expression Levels on Transformants ExpressingImproved Hydroxynitrile Lyases in which the Amino Acid at Position 2 isSubstituted

(1) Preparation of Cell Extracts

Of the transformants expressing the improved hydroxynitrile lyases inwhich the amino acid at position 2 is substituted prepared in (1) and(2) in Example 5, transformants carrying pOXN103V2K, pOXN103V2N,pOXN103V21, pOXN103V2R, pOXN103V2Q, pOXN103V2P, pOXN103V2T, pOXN103V2Y,pOXN103V2L, pOXN103V2M, pOXN103V2S, pOXN103V2E, pOXN103V2A, pOXN103V2Gand pOXN103V2D were cultured in the medium described below (100 ml in500 ml Erlenmeyer flask) at 37° C. for 24 hours.

Medium Composition

Peptone 10 g/L Yeast extract 5 g/L NaCl 10 g/L Ampicillin 50 mg/L IPTG 1mM (final concentration)

As a control, the wild-type hydroxynitrile lyase expressionvector-introduced transformant JM109/pOXN103 obtained in (2) in Example2 was cultured in the same manner.

Cells were harvested from the resultant culture by centrifugation(3,700×g, 10 minutes, 4° C.), washed with 10 mM sodium phosphate buffer(pH 7.0) and suspended in 10 ml of the same buffer. The resultant cellsuspension (1 ml) was disrupted with a sonicator VP-15S (Taitec, Japan)for 3 minutes under the following conditions: output control 4, DUTYCYCLE 40%, PULS, TIMER=B mode 10 s, while ice-cooling. The disruptedcell suspension was used as the total fraction of cell extract.

(2) Analysis of Expression Levels by Polyacrylamide Gel Electrophoresis

The total fraction of cell extract obtained in (1) above was dilutedwith 10 mM sodium phosphate buffer (pH 7.0) to give a concentration ofOD630=12.5 as calculated for the cell density in the culture broth. Thediluted total fraction of cell extract from each E. coli clone was mixedwith an equal amount of polyacrylamide gel electrophoresis sample buffer(0.1M Tris-HCl (pH 6.8), 4% w/v SDS, 12% v/v β-mercaptoethanol, 20% v/vglycerol, trace bromophenol blue), followed by boiling for 5 minutes fordenaturation. 10% polyacrylamide gel was prepared, and the denaturedsample was applied thereto (5 μl per lane) and electrophoresed (FIG. 3).Each hydroxynitrile lyase was observed as an approx. 30 kDa band (thearrow mark in FIG. 3). With JM109/pKK233-2(+Sse) carrying only an emptyvector as background, the thickness of bands corresponding to mutanthydroxynitrile lyases were compared with that of the band of thewild-type hydroxynitrile lyase-expressing transformant JM109/pOXN103using an analysis software Image-Pro Plus Ver 4.5 (Planetron). As aresult, relative values as shown in Table 5 were obtained. It wasdemonstrated that improved hydroxynitrile lyases with greatly enhancedexpression levels can be obtained by substituting the amino acid atposition 2 with another amino acid.

According to the relationship between the amino acid at position 2 andformylmethionine processing, and the N-end rule, it is said that thereis no significant difference in the stability of protein when comparinga protein which has valine at position 2 and a protein which hasisoleucine at position 2. Therefore, the above-described improvementeffect on expression level cannot be explained by those relationship andrule. It is believed that this improvement effect is resulting from athoroughly new principle.

TABLE 5 Relative Ratio of Band Thickness of Improved HydroxynitrileLyases Expression Hydroxynitrile Relative ratio of band thicknessplasmid lyase (%) pOXN103 wild-type 100 pOXN103V2K V2K 736 pOXN103V2NV2N 567 pOXN103V2I V2I 560 pOXN103V2R V2R 446 pOXN103V2Q V2Q 349pOXN103V2P V2P 365 pOXN103V2T V2T 313 pOXN103V2Y V2Y 264 pOXN103V2L V2L294 pOXN103V2M V2M 190 pOXN103V2S V2S 314 pOXN103V2E V2E 155 pOXN103V2AV2A 120 pOXN103V2G V2G 138 pOXN103V2D V2D 105

Example 7 Activity Evaluation on Transformants Expressing ImprovedHydroxynitrile Lyases in which the Amino Acid at Position 2 isSubstituted

Of the transformants expressing the improved hydroxynitrile lyases inwhich the amino acid at position 2 is substituted prepared in (1) and(2) in Example 5, transformants carrying pOXN103V2K, pOXN103V2N,pOXN103V2I, pOXN103V2R and pOXN103V2Q were cultured in the same manneras described in (1) in Example 6 except that they were cultured at 30°C. for 24 hours. As a control, the wild-type hydroxynitrile lyaseexpression vector-introduced transformant JM109/pOXN103 obtained in (2)in Example 2 was cultured in the same manner. From the resultantculture, the total fraction of cell extract was obtained in the samemanner as described in (1) in Example 6 and then centrifuged (10,000×g,5 minutes, 4° C.). The resultant supernatant was collected as thesoluble fraction of cell extract. Using the thus obtained solublefraction of cell extract, hydroxynitrile lyase activity was measuredaccording to the method disclosed in Japanese Unexamined PatentPublication No. 11-508775. Briefly, the activity was measured by tracingthe generation of benzaldehyde from racemic mandelonitrile. The enzymesolution (50 μl) was mixed with 50 mM sodium citrate buffer (pH 5.0)(900 μl). A substrate solution

(100 μl) (37.5 mM racemic mandelonitrile/10 mM sodium citrate buffer (pH3.5)) was added to the above mixture, and the measurement of activitywas started. Increase in absorbance at 280 nm was traced for 5 minutes(as a control, measurement was performed on an enzyme-free substratesolution). 1 U corresponds to the amount of enzyme that catalyzesconversion of racemic mandelonitrile to 1 μmol benzaldehyde per minuteunder the above-described conditions. Further, quantitativedetermination of the protein in the soluble fraction was performed withBio-Rad Protein Assay (Bio-Rad) according to the protocol attachedthereto. As a result, specific activities per mg of protein in cellextract soluble fraction were 22.7 U/mg protein forJM109/pOXN103V2K-derived protein; 8.1 U/mg protein forJM109/pOXN103V2N-derived protein; 5.5 U/mg protein forJM109/pOXN103V2I-derived protein; 7.8 U/mg protein forJM109/pOXN103V2R-derived protein; and 4.5 U/mg protein forJM109/pOXN103V2Q-derived protein. On the other hand, the specificactivity per mg of protein in cell extract soluble fraction of thewild-type hydroxynitrile lyase expression vector-introduced transformantJM109/pOXN103 was 2.7 U/mg protein. Thus, it was confirmed that thespecific activities per protein in cell extract of improvedhydroxynitrile lyases obtained by substituting the amino acid atposition 2 with another amino acid are greatly improved compared to theactivity of protein from the wild-type hydroxynitrile lyase expressionvector-introduced transformant.

Example 8 Random Mutagenesis by Error Prone PCR and Screening

(1) Error Prone PCR (1 st)

Error prone PCR was performed using as a template the E. coli codonwild-type hydroxynitrile lyase expression vector pUMESDsy prepared inExample 4.

With respect to the conditions of the error prone PCR, the quantities ofMgCl₂ and MnCl₂ were increased in the reaction solution. Table 6 showsthe composition of the PCR reaction solution.

TABLE 6 Composition of Error Prone PCR Reaction Solution Template DNA(pUMESDsy) 1 (μl) Primer M13-reverse 1 M13-forward 1 10 × Buffer (forTaq) (not containing MgCl₂) 5 10 mM dATP 1 10 mM dCTP 5 10 mM dGTP 1 10mM dTTP 5 Taq DNA polymerase 2.5 25 mM MgCl₂ 15  5 mM MnCl₂ 10 H₂O 2.5Total 50 (μl)

The sequences of the above-mentioned primers are as follows.

M13-reverse: aacagctatgaccatg (SEQ ID NO: 84)M13-forward: gtaaaacgacggccagt (SEQ ID NO: 85)

The reaction conditions of PCR were as described in Table 7 below.

TABLE 7 Reaction Conditions of Error Prone PCR

The resultant PCR products were extracted from agarose gel using GelExtraction System (VIOGENE).

The amplified DNAs and vector pUC19 were treated with BamHI and SphI,and then electrophoresed. From the electrophoresis gel, the amplifiedDNAs and vector pUC19 were recovered by extraction and ligated. With theligation solution, E. coli JM109 strain was transformed to obtaintransformants. Master plates of mutant colonies were prepared and usedin the subsequent experiment.

(2) Screening of the Mutants Obtained by Error Prone PCR

From the mutants obtained in (1), those with improved activity werescreened. The activity was determined by measuring mandelonitrile(substrate) degrading activity in the cell extract soluble fractionprepared from cultured cells expressing mutants, using the amount ofbenzaldehyde generated as an indicator.

(2-1) Sample Preparation

Samples were prepared using 96-well plates. Into 0.8 ml 96-wellsterilized plates (ABgene), LB medium (containing 80 μg/ml Amp and 0.1mM IPTG) were dispensed at 150 μl/well. Then, mutant colonies wereseeded therein from the master plate. The 96-well plate was subjected toshake culture at 37° C. at 1,200 rpm for 12 hours with BioShaker(M-BR-024, TAITEC). After cultivation, the culture broth was centrifuged(5,000 rpm, 10 min, 4° C., himac CR20, rotor R6S; Hitachi) to harvestthe cells. After removal of the supernatant, the plate was placed upsidedown on newspaper to remove the medium as much as possible. Theresultant cells were suspended in 100 μl of 0.85% NaCl using BioShaker.Then, this suspension was transferred into a 96-well U-bottom plate(Corning). Subsequently, the cells were harvested by centrifugation(4,500 rpm, 10 min, 4° C., himac CR20, rotor R6S; Hitachi) and thesupernatant was removed. The plate was placed upside down on newspaperto remove moisture content as much as possible. To the resultant cells,5 μl of lysozyme solution [10 mg/ml lysozyme (derived from egg white;Seikagaku Corporation), 100 mM KPB (pH 7.0), 10 mM EDTA] was added andsuspended with a TUPLE MIXER (speed 7; IWAKI). The suspension wasincubated at 37° C. for 1 hour to perform lysozyme treatment to therebymake E. coli into protoplast. The resultant E. coli was subjected tofreeze/thaw treatment at −40° C. and 37° C. To the resultant cells, 100μl of a hypotonic solution (10 mM KPB (pH 7.0), 5 mM MgCl₂) was addedfor lysis. The resultant solution was centrifuged (4,500 rpm, 10minutes, 4° C., himac CR20, rotor R6S; HITACHI) to thereby precipitateE. coli genome and cell walls, etc. The resultant supernatant wascollected as a crude enzyme solution and used in the subsequentexperiment.

Alternatively, transformants expressing mutants were cultured, and thecultured cells were washed with phosphate buffer (pH 7) and disrupted bysonication to thereby obtain the total fraction of cell extract. Theliquid containing disrupted cells was centrifuged, and the resultantsupernatant was collected as the soluble fraction of cell extract. Theprecipitate was suspended in phosphate buffer (pH 7.0) in an amountequal to that of supernatant to thereby obtain the insoluble fraction ofcell extract.

(2-2) Activity Measurement

The activity to degrade mandelonitrile (a substrate for hydroxynitrilelyase) was determined by measuring the amount of benzaldehyde generated.The reaction composition is shown in Table 8 below.

TABLE 8 Reaction Composition Final concentration 100 mM Na-citratebuffer (pH 5.4) 100 (μl) 50 mM  10 mM racemic mandelonitrile 80  4 mMEnzyme solution (crude enzyme 10 solution, soluble fraction of cellextract) DIW 10 Total 100 (μl)

For activity measurement, sodium citrate buffer was added to 96-well UVplates (Greiner Bio-One) and its temperature as adjusted to 25° C. Then,the crude enzyme solution was added thereto and suspended by pipetting.Subsequently, racemic mandelonitrile was added thereto, followed bypipetting and shaking. Then, increase in absorbance at 280 nm inwavelength was measured at 25° C. for 10 minutes using a microplatereader (GENios; Tecan Japan). For analysis, LS-PLATE manager 2001 (Win)(Wako Purechemical) was used.

(2-3) Results of Screening

Screening was performed using, as a positive control, pUMESDsy or thesample used as a template for PCR and, as a negative control, pUC19.Specifically, 11 samples showing higher activity than the positivecontrol were selected from approx. 5,000 colonies of the obtainedmutants. Each of these 11 samples was cultured on a 3 ml scale to allowexpression of hydroxynitrile lyase. Then, synthesis activity wasdetermined by measuring production of (S)-mandelonitrile frombenzaldehyde. The standard assay solution contains 300 mM citrate buffer(pH 4.0), 50 mM benzaldehyde and 100 mM cyanide solution in its finalvolume of 0.9 ml. The reaction was immediately started by adding 100 μlof enzyme solution and the reaction solution was incubated at 25° C. for120 minutes. The reaction was terminated by adding 100 μl of sampledreaction solution to 900 μl of organic solvent (hexane:isopropanol=9:1).Then, the supernatant obtained by centrifugation (15,000×g, 10 minutes,4° C.) was assayed by HPLC. The amounts of individual components weremeasured by feeding to CHIRALCEL OJ-H column (Daicel Chemical) at a flowrate of 1.0 ml/minute using hexane:isopropanol=90:10 as a mobile phase.The column temperature was set at 30° C. and absorbance was measured at254 nm. From the resultant standard curves, the amount of individualcomponents were calculated. The amount of enzyme which generates 1 μmolS-mandelonitrile from benzaldehyde per minute under standard assayconditions was defined as 1 unit of enzyme activity.

After the measurement, samples which exhibited remarkably high activitycompared to the positive control were selected. These samples weresubjected to SDS-PAGE to confirm the yield of soluble fraction, andfurther subjected to DNA sequence analysis to confirm the introductionof mutations.

Of the 11 samples used, the sample with the highest activity exhibitedapprox. 10-fold synthesis activity (63.3 U/ml) compared to pUMESDsy; theresults of SDS-PAGE revealed that most of the expressed hydroxynitrilelyase existed in the soluble fraction (Table 9 and FIG. 4). Sequenceanalysis confirmed that this increase in activity is resulted from oneamino acid substitution from His103 to Leu103. The resultant mutantplasmid was designated pUMESDsy-H103L.

(3) Random Mutations by Error Prone PCR (2nd)

Using pUMESDsy-H103L obtained in (2) above as a template, error pronePCR was performed again. The hydroxynitrile lyase synthesis activities(U/ml) of pUMESDsy (used in the 1st error prone PCR as a template),mutant clone pUMESDsy (obtained in the 1st error prone PCR), and randommutant clones 19-E8 and 36-E10 (obtained in the 2nd error prone PCRusing pUMESDsy-H103L as a template) are shown in Table 9 below.

TABLE 9 (S)-HNL Synthesis Activities of Random Mutants 19-E8 36-E10pUMESDsy-H103L pUMESDsy Crude enzyme 74.4 60.1 63.3 4.1 (U/ml)

As a result of the error prone PCR using pUMESDsy-H103L as a template,the resultant clone 19-E8 exhibited further rise in activity (Table 9)and further increase of yield of soluble fraction (FIG. 4). The resultsof sequence analysis revealed that this clone 19-E8 has the followingthree mutations introduced thereinto: His103(cat)→Leu(ctt),Phe125(ttt)→Leu(ctt) and Thr146(acc)→Thr(aca) (two amino acidsubstitutions, three nucleotide mutations). Further, clone 36-E10 whichexhibited less activity increase than the template has the followingthree mutations introduced thereinto: His103(cat)→Leu(ctt),Thr205(acc)→Ser(tcc) and Asp235(gat)→Gly(ggt) (three amino acidsubstitutions, three nucleotide mutations).

Example 9 Introduction of H103L Substitution into Wild-TypeHydroxynitrile Lyase Gene

Plasmid pUMESD-H103L was constructed by mutating the expression vectorpUMESD comprising the plant codon wild-type hydroxynitrile lyase geneprepared in (1) in Example 2 so that the histidine (His, H) at position103 is substituted with leucine (Leu, L). This site-directed mutagenesiswas performed using QuickChange Site-Directed Mutagenesis Kit(STRATAGENE).

PCR reaction conditions are shown in Table 10 below.

TABLE 10 Composition of PCR Reaction Solution 10 × reaction Buffer 5(μl) Template DNA (pUMESD) 1 5′-primer 1.25 3′-primer 1.25 dNTP mix 1H₂O 39.5 Pfu Turbo DNA polymerase 1 Total 50 (μl)

The sequence of the above primer (5′-primer) is as described below.

(SEQ ID NO: 86) OXYN-30: gctggtgttttcctgaattccttattgcc

The sequence of the above primer (3′-primer) is as described below.

(SEQ ID NO: 87) OXYN-31: ggcaataaggaattcaggaaaacaccagc

OXYN-30 (SEQ ID NO: 86) is a sense primer consisting of 29 nucleotidesand has a complementary sequence to OXYN-31. The codon corresponding tothe amino acid at position 103 is CTG and encodes leucine. OXYN-31 (SEQID NO: 87) is an antisense primer consisting of 29 nucleotides and has acomplementary sequence to OXYN-30.

PCR reaction conditions are as shown in Table 11 below.

TABLE 11 PCR Reaction Conditions

In order to introduce H103L mutation into pUMESD, site-directedmutagenesis (single amino acid substitution) was performed by PCR usingthe above primers (OXYN-30 and OXYN-31). One μl of DpnI was added to 50μl of PCR reaction solution, which was then incubated at 37° C. for 1hour. By this treatment, the template DNA was digested and only thoseplasmids into which the mutation had been introduced were obtained.Using the DpnI-treated PCR reaction solution, E. coli JM109 strain wastransformed in the same manner as described in (4) in Example 1.Recombinant vectors were recovered from grown colonies. The recombinantvector comprising the plant codon wild-type hydroxynitrile lyase geneinto which H103L mutation is introduced was designated pUMESD-H103L.

The hydroxynitrile lyase activity of pUMESDsy-H103L obtained in Example8 and the hydroxynitrile lyase activity (synthesis activity) of theabove-described pUMESD-H103L were compared. The measurement ofhydroxynitrile lyase synthesis activity was performed based on themethod as described in Example 8. With respect to cultivation, IPTG wasadded to the culture simultaneously with the start of cultivation togive a final concentration of 0.1 mM. Cells were cultured at 37° C. for12 hours and then harvested.

The hydroxynitrile lyase synthesis activities (U/ml) of the followingfour samples are shown in Table 12: plasmid pUMESDsy comprising the E.coli codon wild-type hydroxynitrile lyase gene, plasmid pUMESDcomprising the plant codon wild-type hydroxynitrile lyase gene, andplasmids pUMESDsy-H103L and pUMESD-H103L obtained by introducing H103Lsubstitution into those plasmids as templates. The results revealed thatthe H103L substitution mutant of plant codon hydroxynitrile lyase gene(pUMESD-H103L) exhibited increase in enzyme activity (Table 12) andyield increase in soluble fraction (FIG. 5) as seen in the correspondingmutant of E. coli codon gene.

TABLE 12 (S)-HNL Synthesis Activities of Amino Acid Substitution MutantsE. coli codon pUMESDsy- Plant codon H103L pUMESDsy pUMESD-H103L pUMESDCrude 56.3 5.7 59.5 4.3 enzyme (U/ml)

Example 10 All Amino Acid Substitution at H103 Residue in E. coli andPlant Codon Wild-Type Hydroxynitrile Lyase Genes

(1) Preparation of H103 Substitution Mutants

Random primers which change the amino acid residue at position 103 into20 essential amino acids were designed in both E. coli and plant codonwild-type hydroxynitrile lyase genes and used for preparing mutants. PCRreaction conditions are shown in Table 13 below.

TABLE 13 Composition of PCR Reaction Solution 10 × reaction Buffer 5(μl) Template DNA (pUMESDsy, pUMESD) 1 5′-primer 1.25 3′-primer 1.25dNTP mix 1 H₂O 39.5 Pfu Turbo DNA polymerase 1 Total 50 (μl)

The sequences for the above primer (5′-primer) are as described below.

H103-20aa-F: ggcgggcgtttttnnsaacagcctgctgcc (SEQ ID NO: 88; for use inamplifying E. coli codon wild-type hydroxynitrile lyase gene)

ME-H103-20aa-F: gcagctggtgttttcnnsaattccttattgccagacaccg (SEQ ID NO: 89;for use in amplifying plant codon wild-type hydroxynitrile lyase gene)

The sequences for the above primer (3′-primer) are as described below.

H103-20aa-R: ggcagcaggctgttsnnaaaaacgcccgcc (SEQ ID NO: 90; for use inamplifying E. coli codon wild-type hydroxynitrile lyase gene)

ME-H103-20aa-R: cggtgtctggcaataaggaattsnngaaaacaccagctgc (SEQ ID NO: 91;for use in amplifying plant codon wild-type hydroxynitrile lyase gene)

In the above primer sequences, n is a, t, g or c.

PCR reaction conditions are as shown in Table 14 below.

TABLE 14 PCR Reaction Conditions

In the same manner as described in Example 9, 20 amino acid substitutionmutants at H103 residue were constructed by site-directed mutagenesisusing QuickChange Site-Directed Mutagenesis Kit (STRATAGENE). Briefly,in order to introduce 20 amino acid substitutions at the H103 residue ofboth pUMESDsy and pUMESD, site-directed mutagenesis (single aminosubstitution) was performed by PCR using the above primers (combinationof H103-20aa-F and H103-20aa-R and combination of ME-H103-20aa-F andME-H103-20aa-R). One μl of DpnI was added to 50 μl of PCR reactionsolution, which was then incubated at 37° C. for 1 hour. By thistreatment, the template DNA was digested and only those plasmids intowhich mutations had been introduced were obtained. With the DpnI-treatedPCR reaction solution, E. coli JM109 strain was transformed. Colonieswere seeded at random in 0.8 ml 96-well sterilized plates (Abgene).After IPTG induction, enzyme solution was prepared by the methoddescribed in (2-1) in Example 8. Subsequently, in the same manner as in(2-2) in Example 8, degradation activity was measured with a microplatereader. When the activity was measured using 96-well plates, templatespUMESDsy and pUMESD and high activity mutants pUMESDsy-H103L andpUMESD-H103L were used as controls. For analysis, LS-PLATE manager 2001(Win) (Wako Purechemical) was used. Plasmids were extracted and purifiedfrom the resultant colonies. Then, their sequences were confirmed by DNAsequence analysis using BigDye Terminator v3.1 Cycle Sequencing Kit(ABI).

(2) Results

By the site-directed mutagenesis using the above-described randomprimers, amino acid substitutions were introduced at random into theH103 residue of both pUMESDsy and pUMESD. DNA sequence analysis revealedthat total 40 types of mutants in which H103 residue was changed to 20amino acids were obtained. The codon of the 103 residue in each aminoacid substitution mutant is shown in Table 15.

TABLE 15 Codons Used in 20 Amino Acid Substitution Mutants at H103Residue of pUMESDsy and pUMESD Amino acid pUMESDsy-derivedpUMESD-derived (one letter abbreviation) (E. coli codon) (plant codon)Ala (A) gcc gcc Val (V) gtc gtc Leu (L) ctc ctg Ile (I) atc atc Pro (P)ccc ccc Phe (F) ttc ttc Trp (W) tgg tgg Met (M) atg atg Gly (G) ggg gggSer (S) agc tcg Thr (T) acc acg Cys (C) tgt tgc Gln (Q) cag cag Asn (N)aac aac Tyr (Y) tac tac Lys (K) aag aag Arg (R) cgc cgc His (H) cat cacAsp (D) gac gac Glu (E) gag gaa

E. coli JM109/pUMESDsy-H103-20aa and JM109/pUMESD-H103-20aa (which aretransformants expressing the resultant mutants) (here, “-20aa”represents one letter abbreviation of the amino acid which replacedH103; for example, when H103 was substituted with leucine, it isexpressed as JM109/pUMESD-H103L) were cultured in LB+Amp (80 μg/ml)medium at 37° C. Simultaneously with the start of the cultivation, IPTGwas added at a final concentration of 0.1 mM. Cells were cultured at 37°C. for 12 hours to allow expression of large quantities ofhydroxynitrile lyases. After 12 hours, cells were harvested in 2.4 mlaliquots, washed with physiological saline, and suspended in 800 μl of10 mM KPB (pH 7.0). Synthesis activity was measured by cell reaction.The results in E. coli codon mutants are shown in FIG. 6(A) and theresults in plant codon mutants are shown in FIG. 6(B). Higher activitythan the wild-type enzyme was confirmed in H103A, H103V, H103I, H103M,H103S, H103T, H103C, H103W and H103Q in addition to H103L. Since theresults are almost equal in both codons, it was confirmed that theeffect of H103 mutation does not depend on the type of codon.

Example 11 Preparation of Transformants Expressing Combined TypeImproved Hydroxynitrile Lyases in which Amino Acids at Positions 2 and103 are Substituted

Based on pOXN103 prepared in (2) in Example 2 and pOXN103V2I prepared in(1) in Example 5, mutants in which the histidine (His; H) at position103 is substituted with leucine (Leu; L) were prepared in the samemanner as in Example 9 using QuickChange Site-Directed Mutagenesis Kit(STRATAGENE). As primers for introducing mutations, OXYN-30 (SEQ ID NO:86) and OXYN-31 (SEQ ID NO: 87) used in Example 9 were used.

According to the protocol attached to the Kit, PCR reaction and DpnItreatment were performed. Subsequently, E. coli JM109 strain wastransformed. Plasmid DNA was recovered from grown colonies. Expressionvectors comprising a gene encoding hydroxynitrile lyase in which theamino acid at position 103 is substituted with leucine were designatedpOXN103H103L (H103L mutation was introduced into pOXN103) andpOXN103V2I+H103L (H103L mutation was introduced into pOXN103V2I).

Example 12 Evaluation of Recombinants Expressing Combined Type ImprovedHydroxynitrile Lyases in which Amino Acids at Positions 2 and 103 areSubstituted

(1) Experimental Method

(1-1) Plasmids and Cell Strains Used

E. coli JM109 strain and C600 strain were transformed with pOXN103(plant codon wild-type hydroxynitrile lyase expression vector) obtainedin (2) in Example 2, pOXN103V2I (plant codon V2I hydroxynitrile lyaseexpression vector) obtained in (1) in Example 5, pOXN103H103L (plantcodon H103L hydroxynitrile lyase expression vector) and pOXN103V2I+H103L(plant codon V2I+H103L hydroxynitrile lyase expression vector) bothobtained in Example 11, in the same manner as described in (4) inExample 1. As a result, transformants JM109/pOXN103, JM109/pOXN103V2I,JM109/pOXN103H103L, JM109/pOXN103V2I+H103L, C600/pOXN103V2I andC600/pOXN103V2I+H103L were obtained.

(1-2) Cultivation Conditions

(1-2-1) Flask Culture Evaluation

Colonies from transformants JM109/pOXN103, JM109/pOXN103V2I,JM109/pOXN103H103L and JM109/pOXN103V2I+H103L were flask-cultured in 100ml of LBAmp medium (containing 0 or 1 mM IPTG) in 500 ml flasks at 30°C. or 37° C. under shaking (210 rpm).

(1-2-2) Jar Culture Evaluation

Colonies from transformants C600/pOXN103V2I and C600/pOXN103V2I+H103Lwere precultured in the medium described below (100 ml in 500 mlErlenmeyer flasks) at 30° C. for 12 hours.

Composition of Preculture Medium (pH 7.2):

Polypeptone N (20 g/L), yeast extract (5 g/L), KH₂PO₄ (1.5 g/L),ampicillin (0.1 g/L)

The rotational speed was 210 rpm.

The resultant preculture (20 ml) was seeded in the main culture mediumdescribed below (2 L in 3 L jar fermenters) and main-cultured at 37° C.or 25° C. for 20-52 hours.

Composition of Main Culture Medium

Polypeptone N  20 g/L Yeast extract   5 g/L KH₂PO₄ 1.5 g/L MgSO₄•7H₂O0.5 g/L MnSO₄•5H₂O 0.2 g/L ZnSO₄•7H₂O 0.02 g/L  CaCl₂•2H₂O 0.02 g/L Pluronic L-61 0.5 g/L Fructose  40 g/L Ampicillin 0.1 g/L

The rotational speed was 750 rpm; air flow rate was 2 L/min; internalpressure was ordinary pressure; pH was controlled at 6.8-7.2 (with 3NNaOH and 5N H₂SO₄). During the culture, sampling was performed from timeto time, followed by measurement of cell density (OD630) andhydroxynitrile lyase degradation activity.

(1-3) Sample Preparation

Measurement of degradation activity was performed as described below.Cells were harvested from the sampled culture broth by centrifugation(3,700×g, 10 minutes, 4° C.), washed with 10 mM sodium phosphate buffer(pH 7.0) or phosphate buffer (pH 7) and suspended in 10 ml of the samebuffer. The resultant cell suspension (1 ml) was disrupted with asonicator VP-15S (Taitec, Japan) for 3 minutes under the followingconditions: output control 4, DUTY CYCLE 40%, PULS, TIMER=B mode 10 s,while ice-cooling. The disrupted cell suspension was collected as thetotal fraction of cell extract. The liquid contacting disrupted cell wascentrifuged (10,000×g, 5 minutes, 4° C.), and the resultant supernatantwas collected as the soluble fraction of cell extract. The precipitatewas suspended in phosphate buffer (pH 7) in an amount equal to that ofthe supernatant, to thereby obtain the insoluble fraction of cellextract.

(1-4) Activity Measurement

Using the soluble fraction of cell extract obtained above,hydroxynitrile lyase activity was measured. Briefly, activity wascalculated by optically detecting (at 280 nm in wavelength) and tracingthe activity to degrade a substrate racemic mandelonitrile (=generationof benzaldehyde).

Enzyme solution (50 μl) was mixed with 900 μl of 50 mM sodium citratebuffer (pH 5.0). A substrate solution (100 μl) (37.5 mM racemicmandelonitrile/10 mM sodium citrate buffer (pH 3.5) freshly preparedeach time) was added to the above mixture to start the activitymeasurement. Increase in absorbance at 280 nm was traced for 5 minutes(as a control, enzyme-free substrate solution was used). One unit (1 U)corresponds to the amount of enzyme which catalyzes conversion of 1 μmolbenzaldehyde from racemic mandelonitrile per minute underabove-described conditions.

(1-5) SDS-PAGE

SDS-PAGE was performed with 10% polyacrylamide gel (AA:Bis=38:2) andTris-Glycine electrophoresis buffer.

(2) Evaluation of Flask Culture of Recombinants into which Amino AcidSubstitutions at Positions 2 and 103 are Introduced JM109/pOXN103,JM109/pOXN103V2I, JM109/pOXN103H103L and JM109/pOXN103V2I+H103L obtainedin (1-1) above were flask-cultured at 30° C. and 37° C. The culture wasperformed in 1 mM (final concentration) IPTG-added LB Amp medium (100ml) (LB medium containing 100 mg/L ampicillin) with a rotational speedof 210 rpm. Individual fractions of cell extract were adjusted to give aconcentration of OD12.5 and analyzed by SDS-PAGE and the degradationactivity of soluble fraction was measured. The results are shown in FIG.7. When H103L mutation alone was introduced into wild-type pOXN103(pOXN103H103L), specific activity per total protein was not greatlychanged at 30° C. but increased about 4 times at 37° C. (FIG. 7;1.15→4.12 U/mg protein). It was confirmed that increase in the ratio ofsoluble enzyme and enhancement of expression level per se alsocontributed to this increase in activity.

Further, when the amino acid mutation H103L at position 103 and theamino acid mutation V2I (pOXN103V2I) at position 2 are combined(pOXN103V2I+H103L), specific activity increased about twice at 30° C.(FIG. 7; 4.18→8.17 U/mg protein) and increased about 10 times at 37° C.(FIG. 7; 1.55→14.4 U/mg protein).

(3) Evaluation of Jar Culture of Recombinants into which Amino AcidSubstitutions at Positions 2 and 103 are Introduced

E. coli C600 strain transformants prepared with pOXN103V2I andpOXN103V2I+H103L were cultured in 3 Ljar fermenters.

The cultivation results are shown in FIG. 8. When C600/pOXN103V2I wascultured at 37° C., almost no activity (specific activity, liquidactivity) was expressed, but C600/pOXN103V2I+H103L showed about 10 timesactivity (specific activity, liquid activity) compared to the former.Here, specific activity (U/mg DC) was calculated by measuring thedegradation activity per ml of cell suspension, and dividing thisactivity value by dry cell mg weight concentration (mg DC/ml) calculatedfrom the cell density OD630 of cell suspension (coefficient was 0.4).Further, liquid activity per culture broth (U/ml) was determined bymultiplying specific activity (U/mg DC) by cell density of culture broth(mg DC/ml; calculated from cell density OD630 using coefficient=0.4).C600/pOXN103V2I+H103L achieved the activity (specific activity, liquidactivity) which C600/pOXN103V2I achieved only after being cultured 50hours or more at 25° C., in 20 hours (37° C.) which is less than half ofthe cultivation time of the former.

Further, it was recognized that the ratio of soluble fraction inC600/pOXN103V2I+H103L was increased more than the results seen in flasklevels (FIG. 8; SDS-PAGE).

From the present Example, activity increase in 37° C. culture wasconfirmed as a result of introduction of the amino acid mutation atposition 103 (H103L) into plant codon hydroxynitrile lyase gene, in thesame manner as confirmed in E. coli codon gene. When plant codonV2I+H103L transformant was cultured at 37° C. in 3 L jar fermenters,more than 10 times activity was obtained compared to 37° C. culture ofcontrol (plant codon V2I). It was confirmed that a combination of aminoacid mutations at position 2 and position 103 causes more synergisticeffect on activity improvement.

Example 13 Influence of the Codon in H103 Residue

Among codons which encode the same amino acid, there are the mostfrequently used codons in high expression genes and the most frequentlyused codons in all genes. The term “high expression gene” refers to, forexample, a gene with high expression level in a great number of E. colispecies. Plasmid pUMESDsy prepared in Example 4 was synthesized usinglatter codons. Then, respective clones in which the codon correspondingto H103 residue is cat or cac were prepared, and hydroxynitrile lyaseactivities and expression levels thereof were examined.

(1) Preparation of Substitution Mutants

Random primers in the codon encoding the His at position 103 weredesigned in pUMESDsy and mutants were prepared. PCR reaction conditionsare shown in Table 16 below.

TABLE 16 Composition of PCR Reaction Solution 10 × reaction Buffer 5(μl) Template DNA (pUMESDsy) 1 5′-primer (10 pmol/μl) 1.25 3′-primer (10pmol/μl) 1.25 dNTP mix 1 H₂O 39.5 Pfu Turbo DNA polymerase 1 Total 50(μl)

The sequence of the above primer (5′-primer) is as described below.

(SEQ ID NO: 88) H103-20aa-F: ggcgggcgtttttnnsaacagcctgctgcc

The sequence of the above primer (3′-primer) is as described below.

(SEQ ID NO: 90) H103-20aa-R: ggcagcaggctgttsnnaaaaacgcccgcc

In both primer sequences, n is a, t, g or c and s is g or c.

PCR reaction conditions are as shown in Table 17 below.

TABLE 17 Error prone PCR Reaction Conditions

Site-directed mutations were introduced using QuickChange Site-DirectedMutagenesis Kit (STRATAGENE) under the above-described conditions. Oneμl of DpnI was added to the PCR reaction solution, which was treated at37° C. for 1 hour. After DpnI treatment, E. coli JM109 strain wastransformed with the treated PCR reaction solution. Plasmids wereextracted from colonies, purified and then subjected to DNA sequenceanalysis using BigDye Terminator v3.1 Cycle Sequencing Kit (ABI). Thus,two mutants in which the H103 codon of pUMESDsy is cat and cac,respectively, were obtained. Three ml of LB+Amp (80 μg/ml) was placed insterilized test tubes, in which colonies of JM109/pUMESDsy (H103cac) andJM109/pUMESDsy (H103cat) were suspended separately and cultured at 37°C. overnight under shaking (preculture). Subsequently, LB+Amp (80μg/ml)+IPTG (0.1 mM) was placed in sterilized test tubes, in whichindividual precultures were seeded at 1% and cultured at 37° C.overnight under shaking (main culture). After 12 hours, 1.5 ml of theculture was centrifuged (8,000 rpm, 10 min, 4° C., himac CF15D; Hitachi)to harvest cells. The resultant cells were washed with 0.85% NaCl, andsuspended in 500 μl of 10 mM KPB (pH 7.0) to perform cell reaction.Then, the activity was measured by HPLC analysis using CHIRALCEL OJ-Hcolumn (Daicel Chemical). Further, a part of the resultant cells wassonicated and centrifuged (8,000 rpm, 10 minutes, 4° C., himac CF15D;Hitachi) to obtain the supernatant as the soluble fraction. Further, tothe precipitate, 8M Urea (in 10 mM KPB (pH 7.0)) was added in 1/10amount relative to the amount of the enzyme solution of disrupted cellsand suspended. The resultant suspension was centrifuged (7,000 rpm, 10minutes, 4° C., himac CF15SD; Hitachi) to obtain the supernatant as theinsoluble fraction. Activity measurement was performed on the solublefraction. Quantitative determination of protein and SDS-PAGE wereperformed on both soluble and insoluble fractions.

(2) Influence by Different Codons

The nucleotides of H103 residue of pUMESDsy were converted to two codons(cat and cac) corresponding to His. The resultantJM109/pUMESDsy−H103(cat) and JM109/pUMESDsy-H103(cac) were cultured inLB+Amp (80 μg/ml) at 37° C. Simultaneously with the start of theculture, IPTG was added thereto at a final concentration of 0.1 mM.Cells were cultured at 37° C. for 12 hours to allow expression of largequantities of hydroxynitrile lyases. After 12 hours, cells wereharvested in 1.5 ml aliquots, washed with physiological saline,suspended in 500 μl of 10 mM KPB (pH 7.0) and disrupted by sonication.The liquid containing disrupted cells was centrifuged at a low speed.The resultant supernatant was collected as the soluble fraction(soluble) and used in activity measurement and quantitativedetermination of protein. The precipitate was dissolved in 8 M urea andcentrifuged at a low speed. The resultant supernatant was collected asthe insoluble fraction (insoluble) and used in quantitativedetermination of protein. SDS-PAGE was performed using 10 μg of eachprotein based on the resultant value.

The results are shown in Table 18 and FIG. 9.

TABLE 18 Influence of His Codons on Hydroxynitrile Lyase Activity His(cat) His (cac) Relative activity (U/ml) 2.01 2.47 Specific activity(U/mg) 1.51 1.64

In cell clones where a high frequency codon in E. coli high expressiongenes (cac) and a high frequency codon in all of the genes in E. coli(cat) were used for H103, respectively, no remarkable difference wasrecognized in activity (Table 18) and expression level (FIG. 9).Therefore, it was believed that there is little influence on activityand expression level between the above two codons.

Example 14 Purification of H103M Improved Hydroxynitrile Lyase andConfirmation of Properties

(1) Purification of Wild-Type Hydroxynitrile Lyase and H103M ImprovedHydroxynitrile Lyase

JM109/pUMESD obtained in (1) in Example 2 and JM109/pUMESD-H103Mobtained in (2) in Example 10 were cultured, and wild-typehydroxynitrile lyase and H103M improved hydroxynitrile lyase werepurified from the individual cells, respectively.

(1-1) Cultivation and Harvesting

Three ml of LB+Amp (80 μg/ml) was placed in sterilized test tubes.Colonies from JM109/pUMESD and JM109/pUMESD-H103M were suspended thereinseparately and cultured at 37° C. overnight (preculture).

Each of the resultant precultures was seeded in the following medium at1%.

Medium Composition

Peptone 10 g/L Yeast extract 5 g/L NaCl 10 g/L Ampicillin 80 mg/L IPTG0.1 mM (final concentration)

The liquid volume of the medium was 10 L in total for JM109/pUMESD and 2L in total for JM109/pUMESD-H103M. Cells were cultured at 37° C. for 12hours (main culture). Then, the culture broth was centrifuged (6,000rpm, 10 minutes, 4° C.) to harvest cells. The resultant cells werewashed with 0.7% NaCl and recentrifuged for harvesting. This washingoperation was repeated twice, and then the cells were suspended insonication buffer (50 mM sodium phosphate/citrate buffer (pH 5.4), 1 mMEDTA) in an amount 5 times as much as the cell wet weight to therebyobtain a cell suspension.

(1-2) Cell Disrupting

Using a sonicator (Insonator model 201M (9 kHz); Kubota Shoji), the cellsuspension was disrupted for 20 minutes. Thereafter, the disruptedliquid was centrifuged (15,000 rpm, 10 minutes, 4° C.) to separate intosupernatant and precipitate. The resultant precipitate was suspended inthe sonication buffer and sonicated again. Centrifugation was performedagain (15,000 rpm, 10 minutes, 4° C.) to obtain the supernatant (cellextract).

(1-3) Thermal Treatment

The cell extract obtained in (1-2) above was transferred into anErlenmeyer flask and subjected to thermal treatment at 60° C. for 10minutes in a water bath. After this treatment, the cell extract wascooled rapidly in ice water and centrifuged (12,000 rpm, 10 minutes, 4°C.) to remove denatured protein, to thereby obtain a crude enzymesolution.

(1-4) Ammonium Sulfate Fractionation

The crude enzyme solution obtained in (1-3) above was 45% ammoniumsulfate-saturated and agitated for 30 minutes. The precipitate obtainedby centrifugation (15,000 rpm, 20 minutes, 4° C.) was collected as 0-45%fraction and dissolved in 10 mM KPB (pH 7.0). Subsequently, thesupernatant was 65% ammonium sulfate-saturated and agitated for 30minutes. The precipitate obtained by centrifugation (15,000 rpm, 20minutes, 4° C.) was collected as 45-65% fraction and dissolved in 10 mMKPB (pH 7.0). Further, the supernatant was 90% ammoniumsulfate-saturated and agitated for 30 minutes. The precipitate obtainedby centrifugation (15,000 rpm, 20 minutes, 4° C.) was collected as65-90% fraction and dissolved in 10 mM KPB (pH 7.0). The proteinsolutions of individual fractions were dialyzed with 10 mM KPB (pH 7.0),followed by activity measurement and quantitative determination ofprotein for each fraction. Activity measurement was performed in thesame manner as in (2-3) in Example 8. Quantitative determination ofprotein was performed with Bio-Rad Protein Assay (Bio-Rad) according tothe attached protocol.

(1-5) DEAE-Toyopearl Column Chromatography

DEAE-Toyopearl resin was packed in a column and, after washing,equilibrated with 10 mM KPB (pH 7.0). Then, the enzyme solution afterdialysis obtained in (1-4) above was applied to the column. Afterwashing with 10 mM KPB (pH 7.0), protein was eluted with linearconcentration gradient of 0-0.5 M NaCl in 10 mM KPB (pH 7.0). Activefractions after elution were collected, dialyzed with 10 mM KPB (pH 7.0)and used in the subsequent step.

(1-6) Butyl-Toyopearl Column Chromatography

The active fractions obtained in (1-5) above were 30% ammoniumsulfate-saturated and applied to Butyl-Toyopearl columnspre-equilibrated with 30% ammonium sulfate-saturated 10 mM KPB (pH 7.0).After washing with 30% ammonium sulfate-saturated 10 mM KPB (pH 7.0),protein was eluted with linear concentration gradient of 30-0% ammoniumsulfate-saturated 10 mM KPB (pH 7.0). Active fractions after elutionwere collected, dialyzed with 10 mM KPB (pH 7.0) and used in thesubsequent step.

(1-7) Superdex 200 HR 10/30 Column Chromatography

Superdex 200 HR 10/30 column was washed with degassed milliQ water andequilibrated with 0.2 M NaCl in 10 mM KPB (pH 7.0). The enzyme solutionafter dialysis obtained in (1-6) above was applied to this column.Protein was eluted with 0.2 M NaCl in 10 mM KPB (pH 7.0). Eluted activefractions were collected, dialyzed with 10 mM KPB (pH 7.0) and used inthe subsequent step.

(1-8) MonoQ HR 10/30

MonoQ HR 10/30 was washed with degassed milliQ water, equilibrated with1 M NaCl in 10 mM KPB (pH 7.0), and washed with 10 mM KPB (pH 7.0).Then, the enzyme solution after dialysis obtained in (1-7) above wasapplied thereto. Protein was eluted with linear concentration gradientof 0-1 M NaCl in 10 mM KPB (pH 7.0). Eluted active fractions werecollected and dialyzed with 10 mM KPB (pH 7.0).

(1-9) Results of Purification

The results of purification of H103M improved hydroxynitrile lyase areshown in Table 19. It was possible to purify H103M improvedhydroxynitrile lyase to 9-fold or more purity through theabove-described steps.

TABLE 19 Purification Steps for H103M Improved Hydroxynitrile Lyasetotal specific activity total protein activity Yield fold (U) (mg)(U/mg) (%) purification Cell extract 43577.8 1088.1 40.1 100 1 Heattreatment 28188.8 396.2 71.2 64.7 1.78 (NH₄)₂SO₄: 18118.1 160.1 113.141.6 2.82 45-65% DEAE-Toyopearl 18533.1 103.3 179.4 42.5 4.47Butyl-Toyopearl 4814.3 16.1 298.6 11.0 7.46 Superdex 6579.2 17.3 380.315.1 9.49

The purified wild-type hydroxynitrile lyase and H103M improvedhydroxynitrile lyase were analyzed by SDS-PAGE. Specific activities perprotein were determined for both enzymes as described later, and 0.3 Uper lane was applied. The results of SDS-PAGE are shown in FIG. 10. Itwas confirmed that both enzymes are purified to a single band. Since theband of H103M improved hydroxynitrile lyase seemed thinner than the bandof wild-type hydroxynitrile lyase, it was suggested that the specificactivity per enzyme protein of H103M improved hydroxynitrile lyase isimproved compared to that of wild-type hydroxynitrile lyase. Actually,the results of determination of specific activities per enzyme proteinof both enzymes confirmed that specific activity of H103M improvedhydroxynitrile lyase is enhanced to about 1.5-fold relative to that ofwild-type hydroxynitrile lyase, as shown in Table 20.

TABLE 20 Specific Activities per Enzyme Protein of Wild-Type and H103MImproved Hydroxynitrile Lyases Wild-type hydroxynitrile H103M improvedlyase hydroxynitrile lyase 98.8 U/mg 143.4 U/mg(2) Effect on Activity of Chelating Agents and Metals Added to ReactionSystem

Using purified enzymes of wild-type hydroxynitrile lyase and H103Mimproved hydroxynitrile lyase obtained in (1) in Example 14, effects onsynthetic activity of chelating agents and metals added tomandelonitrile synthesis reaction system were examined. Specifically, achelating agent (EDTA) and various metals (CoCl₂, NiSO₄, MgCl₂, CaCl₂,NaCl, KCl and LiCl) were added at a final concentration of 1 mM or 10mM. Briefly, mandelonitrile synthesis reaction was performed with thereaction composition as shown in Table 21, and the activity was measuredin the same manner as described in (2-3) in Example 8.

TABLE 21 Reaction Composition Final Concentration 1 mM 10 mM 500 mMNa-citrate buffer (pH 4.0) 600 600 Enzyme solution 100 100 1.25Mbenzaldehyde (dissolved in DMSO) 40 40 1M KCN 100 100 100 mM additivesolution 10 100 (EDTA, CoCl₂, NiSO₄, MgCl₂, CaCl₂, NaCl, KCl, LiCl) DIW150 60 Total 1000 μl 1000 μl

The results are shown in Table 22. Table 22 shows relative activitiestaking the activity in a reaction system without any additive as 100%.For each of the additives, the upper row shows the relative activitywhen the relevant additive was added at 1 mM, and the lower row showsthe relative activity when the relevant additive was added at 10 mM.When EDTA and NiSO₄ were added at 1 mM, no considerable decrease inactivity was observed in both the wild-type hydroxynitrile lyase andH103M improved hydroxynitrile lyase. Difference between these enzymeswas also small. On the other hand, when other metals (CoCl₂, MgCl₂,CaCl₂, NaCl, KCl, LiCl) were added, in particular added at 10 mM in thereaction solution, relatively large decrease in activity was observed.Although the activity decrease ratio varied depending on the metaladded, it was confirmed that the activity decrease ratio in H103Mimproved hydroxynitrile lyase was smaller than that in wild-typehydroxynitrile lyase. Thus, it was confirmed that because of H103Mmutation, H103M improved hydroxynitrile lyase has become lesssusceptible to the effect of metals.

TABLE 22 Effect of Metals on Mandelonitrile Synthesis Reaction SystemWild-type H103M improved hydroxynitrile lyase hydroxynitrile lyase EDTA93.9 92.2 83.4 100.4 CoCl₂ 65.4 88.0 26.1 35.0 NiSO₄ 99.6 91.8 68.0 85.4MgCl₂ 72.2 79.2 36.5 39.8 MnCl₂ 67.6 78.6 35.5 38.4 CaCl₂ 73.7 80.4 37.443.0 NaCl 80.7 88.7 46.2 53.1 KCl 76.0 88.2 47.6 50.9 LiCl 86.9 90.548.5 57.2

Example 15 Preparation and Evaluation of Improved Hydroxynitrile Lyasewith Lysine Residue Single Substitution Mutation

(1) Introduction of Site-Directed Single Substitution Mutation intoLysine Residue

Using the expression vector pUMESDsy prepared in Example 4 comprisingthe E. coli colon wild-type hydroxynitrile lyase gene as a template, E.coli colon improved hydroxynitrile lyase genes encoding an improvedhydroxynitrile lyase in which the lysine residue at position 176, 199 or224 is substituted with another amino acid were prepared bysite-directed mutagenesis. The site-directed mutagenesis was performedusing QuickChange Site-Directed Mutagenesis Kit (Stratagene) and primerswhich introduce random mutation into the amino acid at position 176, 199or 224.

PCR reaction conditions are shown in Table 23.

TABLE 23 Composition of PCR Reaction Solution 10 × reaction Buffer 5(μl) Template DNA (pUMESDsy) 1 5′-primer 1.25 3′-primer 1.25 dNTP mix 1H₂O 39.5 Pfu Turbo DNA polymerase 1 Total 50 (μl)

Sequences for the 5′-primer are as described below.

For position 176 mutant: K176-F: (SEQ ID NO: 92)ctggcgaaaatggtgatgcgcnnsggcagcctgtttcagaacgtgc For position 199 mutant:K199-F: (SEQ ID NO: 93)cgaaaaaggctatggcagcattnnsaaagtgtatatttggaccgatcaggFor position 224 mutant: K224-F: (SEQ ID NO: 94)gcgctggcagattgcgaactatnnnccggataaagtgtatcagg

Sequences for the 3′-primer are as described below.

For position 176 mutant: K176-R: (SEQ ID NO: 95)gcacgttctgaaacaggctgccsnngcgcatcaccattttcgccag For position 199 mutant:K199-R: (SEQ ID NO: 96) cctgatcggtccaaatatacactttsnnaatgctgccatagcctttttFor position 224 mutant: K224-R: (SEQ ID NO: 97)cctgatacactttatccggnnnatagttcgcaatctgccagcgc

In the above primer sequences, n is a, t, g or c and s is g or c.

PCR reaction conditions are as shown in Table 24 below.

TABLE 24 PCR Reaction Conditions

One μl of DpnI was added to 50 μl of PCR reaction solution, which wasthen incubated at 37° C. for 1 hour. By this treatment, the template DNAwas digested and only those plasmids into which mutations had beenintroduced were obtained. Using the DpnI-digested PCR reaction solution,E. coli JM109 strain was transformed. Master plates of mutant colonieswere prepared and used in the activity increase screening experimentdescribed below.

(2) Screening of Mutants

From the mutants obtained in (1) above, those which show increasedactivity were screened.

(2-1) Primary Screening

Samples were prepared in 96-well plates. LB medium (containing 80 μg/mlAmp and 0.1 mM IPTG) was dispensed into 0.8 ml 96-well sterilized plates(Abgene) at 150 μl/well. Then, mutant colonies were seeded therein fromthe master plate. The 96-well plate was subjected to shake culture at37° C. at 1,200 rpm for 12 hours with BioShaker (M-BR-024, TAITEC).After cultivation, the culture broth was centrifuged (5,000 rpm, 10 min,4° C., himac CR20, rotor R6S; Hitachi) to harvest the cells. Afterremoval of the supernatant, the plate was placed upside down onnewspaper to remove the medium as much as possible. The resultant cellswere suspended in 100 μl of 0.85% NaCl using BioShaker. Then, thissuspension was transferred into a 96-well U-bottom plate (Corning).Subsequently, the cells were harvested by centrifugation (4,500 rpm, 10min, 4° C., himac CR20, rotor R6S; Hitachi) and the supernatant wasremoved. The plate was placed upside down on newspaper to removemoisture content. To the resultant cells, a lysozyme solution [10 mg/mllysozyme (derived from egg white; Seikagaku Corporation), 100 mM KPB (pH7.0), 10 mM EDTA] was added and suspended with a TUPLE MIXER (speed 7;IWAKI). The suspension was incubated at 37° C. for 1 hour to performlysozyme treatment to thereby make E. coli into protoplast. Theresultant E. coli was subjected to freeze/thaw treatment at −40° C. and37° C. To the resultant cells, 100 μl of a hypotonic solution (10 mM KPB(pH 7.0), 5 mM MgCl₂) was added for lysis. The resultant solution wascentrifuged (4,500 rpm, 10 minutes, 4° C., himac CR20, rotor R6S;Hitachi) to thereby precipitate E. coli genome and cell walls, etc. Theresultant supernatant was collected as a crude enzyme solution and usedin the subsequent activity measurement.

The activity to degrade mandelonitrile (a substrate for hydroxynitrilelyase) was determined by measuring the amount of benzaldehyde generated.The reaction composition is shown in Table 25 below.

TABLE 25 Reaction Composition Final Concentration 100 mM Na-citratebuffer (pH 5.4) 100 (μl) 50 mM  10 mM racemic mandelonitrile 80  4 mMEnzyme solution (crude enzyme 10 solution) DIW 10 Total 100 (μl)

For activity measurement, sodium citrate buffer was added to 96-well UVplates (Greiner Bio-One) and its temperature was adjusted to 25° C.Then, the crude enzyme solution was added thereto and suspended bypipetting. Subsequently, racemic mandelonitrile was added thereto,followed by pipetting and shaking. Then, increase in absorbance at 280nm in wavelength was measured at 25° C. for 10 minutes using amicroplate reader (GENios; Tecan Japan). For analysis, LS-PLATE manager2001 (Win) (Wako Purechemical) was used. Screening was performed usingpUMESDsy as a positive control and pUC19 as a negative control. For eachof the position 176 mutant, position 199 mutant and position 224 mutant,samples showing higher activity than positive control were selected from188 samples. As a result, 12 samples of position K176 mutant, 2 samplesof position K199 mutant and 11 samples of position K224 mutant wereobtained.

(2-2) Secondary Screening

Three ml each of LB+Amp (80 μg/ml) was placed in sterilized test tubes.The 12 samples of position K176 mutant, 2 samples of position K199mutant and 11 samples of position K224 mutant all of which showed higheractivity than positive control in the primary screening; the positivecontrol pUMESDsy and the negative control pUC19 were seeded therein andcultured at 37° C. overnight under shaking (preculture). After 12 hours,1.5 ml of the culture was centrifuged (8,000 rpm, 10 min, 4° C., himacCF15D; Hitachi) to harvest cells. The resultant cells were washed with0.85% NaCl, suspended in 500 μl of 10 mM KPB (pH 7.0), and disrupted.The resultant disrupted cells were centrifuged (8,000 rpm, 10 min, 4°C., himac CF15D; Hitachi) to obtain the supernatant as a solublefraction. Using the thus obtained soluble fraction, activity measurementwas performed. Briefly, production of (S)-mandelonitrile frombenzaldehyde was analyzed by HPLC using a chiral column to therebydetermine the synthesis activity. The standard assay solution contains300 mM citrate buffer (pH 4.0), 50 mM benzaldehyde and 100 mM cyanidesolution in its final volume of 0.9 ml. The reaction was immediatelystarted by adding 100 μl of enzyme solution and the reaction solutionwas incubated at 25° C. for 120 minutes. The reaction was terminated byadding 900 μl of organic solvent (hexane:isopropanol=9:1). Then, thesupernatant obtained by centrifugation (15,000×g, 10 minutes) wasassayed by HPLC. The amounts of individual components were measured byfeeding to CHIRALCEL OJ-H column (Daicel Chemical) at a flow rate of 1.0ml/minute using hexane:isopropanol=90:10 as a mobile phase and measuringabsorbance at 254 nm. From the resultant standard curves, the amounts ofindividual components were calculated. The amount of enzyme whichgenerates 1 μmol S-mandelonitrile from benzaldehyde per minute understandard assay conditions was defined as 1 unit of enzyme activity. Foreach of the position 176 mutant, position 199 mutant and position 224mutant, samples showing higher activity than positive control wereconfirmed. Using soluble fractions from these high activity samples,quantitative determination of protein and SDS-PAGE (10 μg of each sampleprotein) were performed.

(2-3) Results of Screening

As a result of activity measurement, samples with remarkably higheractivity than the positive control were confirmed in each of theposition 176 mutant, position 199 mutant and position 224 mutant.Recombinant vectors were prepared from these samples, and nucleotidesequences thereof were examined in the same manner as described in (4)in Example 1. The results revealed that, in position 176 mutant, thecodon aaa encoding lysine in wild-type hydroxynitrile lyase has beenchanged to ccc that encodes proline (K176P). Likewise, in position 199mutant, the codon aaa encoding lysine in wild-type hydroxynitrile lyasehas been changed to ccc that encodes proline (K199P); and in position224 mutant, the codon aaa encoding lysine in wild-type hydroxynitrilelyase has been changed to cct that encodes proline (K224P).

The activities per soluble protein of K176P, K199P and K224P singlesubstitution mutants are shown in Table 26. K176P showed 2.9-foldactivity compared to positive control pUMESDsy; K199P showed 2.3-foldactivity compared to positive control pUMESDsy; and K224P showed3.3-fold activity compared to positive control pUMESDsy. Further, theresults of SDS-PAGE analysis (FIG. 11) confirmed that the amount ofhydroxynitrile lyase in soluble fraction is increased in any of thesingle substitution mutants K176P, K199P and K224P. These resultsconfirmed that it is possible to improve the expression level andactivity of hydroxynitrile lyase by substituting a lysine residue withanother amino acid, especially proline, in the amino acid sequence of awild-type hydroxynitrile lyase.

TABLE 26 Activities of Lysine Substitution Mutants Sample Hydroxynitrilelyase activity (U/mg-protein) pUMESDsy 1.5 K176P 4.4 K199P 3.4 K224P 4.9K176P × K224P 10.7 K199P × K224P 8.0 K176P × K199P × K224P 11.1

Example 16 Preparation and Evaluation of Improved Hydroxynitrile Lyaseswith Lysine Residue Multiple Substitutions

(1) Preparation of Lysine Residue Multiple Substitution Mutants

E. coli codon improved hydroxynitrile lyase genes encoding multiplesubstitution mutants in which two or three lysine residues aresubstituted were prepared based on the lysine residue singlesubstitution mutants obtained in Example 15. First, using expressionvector pUMESDsy-K224P comprising K224P mutation as a template, a doublemutant in which the lysine residues at positions 176 and 224 aresubstituted with proline residues (K176P×K224P) and a double mutant inwhich the lysine residues at positions 199 and 224 are substituted withproline residues (K199P×K224P) were prepared by site-directedmutagenesis. Further, using expression vector pUMESDsy-K176P×K224Pcomprising the resultant double mutation K176P×K224P as a template, atriple mutant in which all the lysine residues at positions 176, 199 and224 are substituted with proline residues (K176P×K199×K224P) wasprepared. As described in (1) in Example 15, site-directed mutagenesiswas preformed using QuickChange Site-Directed Mutagenesis Kit(Stratagene).

PCR reaction conditions are shown in Table 27 below.

TABLE 27 Composition of PCR Reaction Solution 10 × reaction Buffer 5(μl) Template DNA 1 5′-primer 1.25 3′-primer 1.25 dNTP mix 1 H₂O 39.5Pfu Turbo DNA polymerase 1 Total 50 (μl)

The template DNA above is pUMESDsy-K224P when preparing K176P×K224Pmutant and K199P×K224P mutant; and the template DNA above ispUMESDsy-K176P×K224P when preparing mutation K176P×K199P×K224P.

Sequences for 5′-primer are as described below.

For preparing K176P×K224P mutant:

K176P-F: (SEQ ID NO: 98) ctggcgaaaatggtgatgcgcccnggcagcctgtttcagaacgtgcFor preparing K199P×K224P mutant and K176P×K199P×K224P mutant:

K199P-F: (SEQ ID NO: 99)cgaaaaaggctatggcagcattccnaaagtgtatatttggaccgatcagg

Sequences for 3′-primer are as described below.

For preparing K176P×K224P mutant:

K176P-R: (SEQ ID NO: 100) gcacgttctgaaacaggctgccngggcgcatcaccattttcgccagFor preparing K199P×K224P mutant and K176P×K199P×K224P mutant:

K199P-R: (SEQ ID NO: 101)cctgatcggtccaaatatacactttnggaatgctgccatagccttttt

In the above primer sequences, n is a, t, g or c. The codon specifyingproline is cct, ccc, cca or ccg. The mutation introduction primersdescribed above are designed so that any of the proline codons alwayscomes at the mutation site of interest (in 5′-primer, ccn; in 3′-primer,ngg).

PCR reaction conditions are the same as described in (1) in Example 15.

One μl of DpnI was added to 50 μl of PCR reaction solution, which wasthen incubated at 37° C. for 1 hour. By this treatment, the template DNAwas digested and only those plasmids into which mutations had beenintroduced were obtained. With the DpnI-treated PCR reaction solution,E. coli JM109 strain was transformed in the same manner as describe in(4) in Example 1. Recombinant vectors were prepared from the resultanttransformants, and analysis of the nucleotide sequences thereof wereperformed in the same manner as described in (3) in Example 1. As aresult, it was confirmed that the double mutant in which the lysineresidues at positions 176 and 224 are substituted with proline(K176P×K224P), the double mutant in which the lysine residues atpositions 199 and 224 are substituted with proline (K199P×K224P), andthe triple mutant in which all the lysine residues at positions 176, 199and 224 are substituted with proline (K176P×K199×K224P) were preparedcorrectly.

(2) Activities and Expression Levels of Lysine Residue MultipleSubstitution Mutants

The resultant lysine residue multiple substitution mutants (K176P×K224P,K199P×K224P and K176P×K199P×K224P) were cultured in the same manner asdescribed in (2-3) in Example 15. After preparation of solublefractions, activity measurement, quantitative determination of proteinand SDS-PAGE (10 μg of each sample protein) were performed. The resultsof activity measurement are shown in Table 26. K176P×K224P showed7.1-fold activity compared to positive control pUMESDsy; K199P×K224Pshowed 5.3-fold activity compared to positive control pUMESDsy; andK176P×K199P×K224P showed 7.4-fold activity compared to positive controlpUMESDsy. Further, as a result of SDS-PAGE analysis (FIG. 11), it wasconfirmed that the amount of hydroxynitrile lyase in soluble fraction isincreased more in any of the multiple substitution mutants ofK176P×K224P, K199P×K224P and K176P×K199P×K224P than in theabove-described single substitution mutants. These results confirmedthat substitution of a plurality of lysine residues with other aminoacids, especially proline, in the amino acid sequence of a wild-typehydroxynitrile lyase is capable of improving hydroxynitrile lyaseexpression level per cell and activity remarkably.

Example 17 Preparation and Evaluation of Combined Type ImprovedHydroxynitrile Lyases in which Histidine Residue at Position 103 andLysine Residue(s) are Substituted

(1) Preparation of Combined Type Mutants in which Histidine Residue atPosition 103 and Lysine Residue(s) Are Substituted

Combined type mutants were prepared by introducing H103L mutation(histidine residue at position 103 is substituted with leucine) into thelysine residue single substitution or multiple substitution mutantsobtained in Examples 15 and 16. As templates, pUMESDsy−K176P, −K199P,−K224P (these are lysine residue single substitution mutants) obtainedin Example 15; and −K176P×K224P, −K199P×K224P (these are lysine residuedouble substitution mutants) and K176P×K199P×K224P (lysine residuetriple substitution mutant) obtained in Example 16 were used. In thesame manner as described in Example 9, mutants of interest were preparedusing Quick Change Site-Directed Mutagenesis Kit (STRATAGENE). Asmutation introduction primers, OXYN-30 (SEQ ID NO: 86) and OXYN-31 (SEQID NO: 87) used in Example 9 were used.

According to the protocol attached to the Kit, PCR reaction wasperformed and the reaction solution was treated with DpnI. E. coli JM109strain was transformed, and plasmid DNA was recovered from growncolonies. As a result, hydroxynitrile lyase mutants in which one tothree lysine residues are substituted and yet the amino acid at position103 is substituted with leucine were obtained; K176P+H103L, K199P+H103L,K224P+H103L (these are lysine residue single substitution+H103Lmutants), K176P×K224P+H103L, K199P×K224P+H103L (these are lysine residuedouble substitution+H103L mutants) and K176P×K199P×K224P+H103L.

(2) Evaluation of Combined Type Mutants in which Histidine Residue atPosition 103 and Lysine Residue(s) are Substituted

Each transformant obtained in (1) above was cultured in 3 ml of LBmedium at 37° C. for 12 hours. Simultaneously with the start of theculture, IPTG was added thereto at a final concentration of 0.1 mM.After 12 hours, cells were harvested, washed with physiological salineand suspended in ⅓ volume of 10 mM KPB (pH 7.0). Activity measurementwas performed by cell reaction. From the resultant value, activity perculture broth was calculated. The results are shown in Table 28. Aremarkable improvement in activity compared to the wild-type wasconfirmed in any of the combined type mutants.

TABLE 28 Activities of Combined Type Mutants in which Histidine Residueat Position 103 and Lysine Residue(s) Are Substituted U/ml culturepUMESDsy 9.0 pUMESDsy-K176P × H103L 99.0 pUMESDsy-K199P × H103L 120.9pUMESDsy-K224P × H103L 98.1 pUMESDsy-K176P × K224P × H103L 109.5pUMESDsy-K199P × K224P × H103L 108.0 pUMESDsy-K176P × K199P × K224P ×H103L 96.9

Example 18 Synthesis of Cyanohydrin and Hydroxycarboxylic Acid withImproved Hydroxynitrile Lyase

(1) Synthesis of Cyanohydrin

C600/pOXN103V2I jar culture (40 ml) obtained in (3) in Example 12 wascentrifuged (3,700×g, 10 minutes, 4° C.). After removal of 35 ml ofsupernatant, the remaining cells and culture broth were resuspended. Theresultant cell suspension was disrupted with a sonicator VP-15S (Taitec,Japan) under the following conditions: output control 4, DUTY CYCLE 40%,PULS, TIMER=B mode 10 s, while ice-cooling. Three minutes disrupting wasrepeated 5 times. The liquid containing disrupted cell suspension wascentrifuged again (10,000×g, 5 minutes, 4° C.), and the resultantsupernatant was collected as an enzyme solution of improvedhydroxynitrile lyase. The degradation activity of this enzyme solutionwas calculated in the same manner as described in (1-4) in Example 12.The activity of this enzyme solution was 1171 U/ml. The enzyme solution(3.7 g) (corresponding to 6450 U) was mixed with t-butylmethylether(175.1 g). While maintaining the reaction system at 15-18° C. andagitating sufficiently, HCN (47.6 g) and benzaldehyde (124.6 g) wereadded dropwise continuously over about 4 hours. After completion of thedropping, the reaction system was maintained at 15-18° C. for one hourand agitated sufficiently. After completion of the reaction, thereaction solution was analyzed by HPLC in the same manner as describedin (2-3) in Example 8. As a result, the concentration of mandelonitrilewas 45% by weight and the optical purity thereof was 98% ee S-enantiomerexcess.

(2) Synthesis of Hydroxycarboxylic Acid

While agitating at 30-35° C. for 16 hours, the mandelonitrile solution(131 g) obtained in (1) above was added dropwise to 35% hydrochloricacid (147 g). This reaction solution was a slurry. The reaction solutionafter 16 hours agitation was analyzed by HPLC. As a result,mandelonitrile was not detected, and mandelamide and mandelic acid werepresent in a mixed state. To the total volume of the reaction solutionafter 16 hours agitation, 320 g of water was added and agitated at 75°C. for 2 hours for hydrolysis. This reaction solution was homogeneous.HPLC analysis of this reaction solution revealed that mandelonitrile andmandelamide were not detected, and that the concentration of mandelicacid was 21% and the optical purity thereof was 98% ee S-enantiomerexcess.

INDUSTRIAL APPLICABILITY

According to the present invention, improved hydroxynitrile lyases inwhich amino acids in a wild-type hydroxynitrile lyase are substituted;and genes encoding the same can be obtained. The improved hydroxynitrilelyase gene of the present invention includes not only those genesobtained by introducing mutations into wild-type genes, but also thosegenes obtained by introducing mutations into host codon-typehydroxynitrile lyase genes which have been mutated according to codonusage of the relevant host.

Further, in a transformant obtained by transducing the gene of thepresent invention into a host, hydroxynitrile lyase activity pertransformant can be improved greatly. Therefore, such a transformant iscapable of producing the improved hydroxynitrile lyase in a largequantity and efficiently. Further, with the improved hydroxynitrilelyase of the present invention, it is possible to produce opticallyactive cyanohydrins and optically active hydroxycarboxylic acidefficiently.

SEQUENCE LISTING FREE TEXT

SEQ ID NOS: 3-101 Synthetic DNAs

1. An isolated or purified modified hydroxynitrile lyase that is atleast 90% homologous to the wild-type hydroxynitrile lyase of Manihotescuela of SEQ ID NO: 1 or at least 90% homologous to the wild-typehydroxynitrile lyase of Hevea brasiliensis of SEQ ID NO: 102, and thathas a substitution of the histidine residue corresponding to thehistidine residue at position 103 of SEQ ID NO: 1 or SEQ ID NO: 102 witha non-histidine amino acid residue.
 2. The modified hydroxynitrile lyaseof claim 1 that is at least 90% homologous to the wild-typehydroxynitrile lyase of Manihot escuela of SEQ ID NO:
 1. 3. The modifiedhydroxynitrile lyase of claim 1 that is at least 90% homologous to thewild-type hydroxynitrile lyase of Hevea brasiliensis of SEQ ID NO: 102.4. The modified hydroxynitrile lyase of claim 1, wherein the amino acidresidue corresponding to position 2 of the amino acid sequence of SEQ IDNO: 1 is substituted with a different amino acid residue than in SEQ IDNO:
 1. 5. The modified hydroxynitrile lyase of claim 1, wherein thehistidine residue corresponding to position 103 in the amino acidsequence of SEQ ID NO: 1 or SEQ ID NO: 102 is substituted with an aminoacid selected from the group consisting of methionine, leucine,isoleucine, and valine.
 6. The modified hydroxynitrile lyase of claim 1,wherein the histidine residue corresponding to position 103 of SEQ IDNO: 1 or SEQ ID NO: 102 is substituted with an amino acid selected fromthe group consisting of cysteine, glutamine, serine, threonine, alanine,and tryptophan.
 7. The modified hydroxynitrile lyase of claim 1, whereinat least one lysine residue corresponding to a lysine residue of theamino acid sequence of SEQ ID NO: 1 is substituted with an amino acidresidue other than lysine.
 8. The modified hydroxynitrile lyase of claim1, wherein the amino acid residue corresponding to position 2 of SEQ IDNO: 102 is substituted with different amino acid residue than that inSEQ ID NO:
 102. 9. The modified hydroxynitrile lyase of claim 1, whereinthe amino acid residue corresponding to position 2 of SEQ ID NO: 1 andat least one lysine residue corresponding to a lysine residue of SEQ IDNO: 1 are substituted with different amino acid residues than those inSEQ ID NO:
 1. 10. The modified hydroxynitrile lyase of claim 1, whereinat least one lysine residue corresponding to a lysine residue of theamino acid sequence of SEQ ID NO: 102 is substituted with a differentamino acid residue than than that in SEQ ID NO:
 102. 11. The modifiedhydroxynitrile lyase of claim 1, wherein the amino acid residuecorresponding to position 2 of SEQ ID NO: 102 and at least one lysineresidue corresponding to a lysine residue of SEQ ID NO: 102 aresubstituted with different amino acid residues than those in SEQ ID NO:102.
 12. The modified hydroxynitrile lyase of claim 1, wherein theposition corresponding to position 103 of SEQ ID NO: 1 or 102 issubstituted with a neutral amino acid.
 13. The modified hydroxynitrilelyase of claim 1, wherein the position corresponding to position 103 ofSEQ ID NO: 1 or 102 is substituted with an amino acid selected from thegroup consisting of ala, asn, asp, cys, gln, glu, gly, iso, leu, lys,met, phe, pro, ser, thr, trp, tyr and val.
 14. The hydroxynitrile lyaseof claim 1, wherein at least one lysine residue present in a regioncorresponding to positions 175 to 224 of the amino acid sequence of SEQID NO: 1 or SEQ ID NO: 102 is substituted with a different amino acidthan those in SEQ ID NO: 1 or SEQ ID NO:
 102. 15. The hydroxynitrilelyase of claim 1, wherein at least one lysine residue present in aregion corresponding to positions 175 to 224 of the amino acid sequenceof SEQ ID NO: 1 or SEQ ID NO: 102 is substituted with an amino acidhaving one or both of properties (a) and (b): (a) an amino acidcontaining one or two nitrogen atoms in its molecule selected from thegroup consisting of ala, asn, asp, cys, gln, glu, gly, iso, leu, met,phe, pro, ser, thr, trp, tyr and val; (b) a neutral amino acid.
 16. Thehydroxynitrile lyase of claim 1, wherein at least one lysine residuepresent in a region corresponding to positions 175 to 224 of the aminoacid sequence of SEQ ID NO: 1 or SEQ ID NO: 102 is substituted withproline.
 17. The hydroxynitrile lyase of claim 1, wherein at least onelysine residue selected from the group consisting of the lysine residuescorresponding to positions 176, 199, and 224 in the amino acid sequenceof SEQ ID NO: 1 is substituted with a different amino acid than that inSEQ ID NO:
 1. 18. The hydroxynitrile lyase of claim 1, wherein at leastone lysine residue selected from the group consisting of the lysineresidues corresponding to positions 175, 198, and 223 in the amino acidsequence of SEQ ID NO: 102 is substituted with a different amino acidthan that in SEQ ID NO:
 102. 19. The modified hydroxynitrile lyase ofclaim 1 that has a higher specific activity than the wild-typehydroxynitrile lyase of SEQ ID NO: 1 or SEQ ID NO:
 102. 20. The modifiedhydroxynitrile lyase of claim 1 that has a higher specific activity tocatalyze a reaction producing a cyanohydrin from either ketone or fromaldehyde and a cyanide compound, as well as the ability to catalyze areverse reaction thereof, than the wild-type hydroxynitrile lyase of SEQID NO: 1 or SEQ ID NO:
 102. 21. A composition comprising the isolated orpurified modified hydroxynitrile lyase of claim
 1. 22. An isolated orpurified polynucleotide encoding the hydroxynitrile lyase of claim 1.23. A recombinant vector comprising the polynucleotide of claim
 22. 24.A transformant obtained by introducing the recombinant vector of claim23 into a host.
 25. A culture obtained by culturing the transformant ofclaim
 24. 26. A modified hydroxynitrile lyase of claim 1 that isrecombinantly produced by a host cell transformed with a polynucleotideencoding said lyase.
 27. A method for producing a hydroxynitrile lyase,comprising recovering the improved hydroxynitrile lyase from the cultureof claim
 25. 28. A method for producing a cyanohydrin, comprising:treating a ketone compound or aldehyde compound, and a cyanide compoundwith the culture of claim 25, and recovering the cyanohydrin from thetreated culture.
 29. A method for producing a cyanohydrin, comprising:treating a ketone compound or an aldehyde compounds, and a cyanidecompound with the modified hydroxynitrile lyase of claim 1, andrecovering the cyanohydrin from the treated culture.
 30. A method forproducing a hydroxycarboxylic acid, comprising hydrolyzing thecyanohydrin obtained by the method of claim 28.